Yohimbine Enhances the Effect of Sildenafil on Erectile Process in Rats

ORIGINAL ARTICLE Yohimbine enhances the effect of sildenafil on erectile process in rats

AM Senbel1 and T Mostafa2

1Pharmacology Department, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt and 2Andrology and Sexology Department, Faculty of Medicine, Cairo University, Cairo, Egypt

Combining the centrally acting drug yohimbine with the peripheral conditioner sildenafil might be an approach to erectile dysfunction cases in which sildenafil alone failed. This work aimed to investigate the effect of yohimbine on sildenafil-induced facilitation of erectile process. Erectile responses to electrical stimulation of the cavernous nerve in anesthetized male rats were recorded. Intracavernosal pressure/systemic arterial pressure (ICP/SAP) was calculated, 1 and 5 min after intravenous administration of sildenafil, yohimbine or a combination of both. Changes in sexual arousal and copulatory performance indices were compared before and after these injections using behavioral mating experiments. It was shown that systemic administration of sildenafil produced a significant increase in ICP/SAP than control at doses X10 lmol kgÀ1. Yohimbine alone failed to potentiate erectile responses but yohimbine (1 lmol kgÀ1) significantly potentiated the effect of sildenafil 1–10 lmol kgÀ1 and 1 mmol kgÀ1, 1 and 5 min after injection. Potentiation of ICP/SAP induced by their combination was greater than the sum of the effects of the corresponding doses of either drug at the same time interval. A nonsignificant additional decrease in SAP than sildenafil- induced was observed if administered with yohimbine. Addition of sildenafil to yohimbine significantly enhanced the effect of the latter on intromission frequency, intercopulatory interval and the number of ejaculations per session. It is concluded that yohimbine may enhance and prolong the effect of sildenafil on erectile process without additional hypotension. Sildenafil may enhance the central effects of yohimbine on erection; it amplifies the effect of yohimbine on male copulatory performance but not on sexual motivation. The potential beneficial effect of the combination was found to be more pronounced on the central component than on the peripheral component of the erectile process. International Journal of Impotence Research (2008) 20, 409–417; doi:10.1038/sj.ijir.3901630; published online 17 April 2008

Keywords: erectile dysfunction; sildenafil; yohimbine; phosphodiesterase inhibitor; corpus cavernosum

Introduction agents, including the PDE5 inhibitors, phentola- 1,2 mine, yohimbine, L-arginine and so on. Sexual behavior and erectile function are influenced Introduction of sildenafil citrate (Viagra Pfizer by emotional and cognitive functions. At the central Inc., New York, NY, USA) in 1998, has been an nervous system level hypothalamic and limbic advancement in improving erectile function and systems are responsible for central erectogenic enabling successful sexual intercourse in men with signals facilitating spinal cord pathways, which broad spectrum ED.3,4 Yohimbine is an indole lead to erection via peripheral autonomic nerves.1 alkaloid whose aphrodisiac activity may be Current strategies for the pharmacological treatment mediated through a combination of central nervous of erectile dysfunction (ED) favor the use of oral system effects and peripheral effects including blockade of pre- and postsynaptic a2-adrenergic receptors.5–7 Placebo-controlled studies have suggested the effectiveness of yohimbine in treating ED Correspondence: Professor Dr T Mostafa, Andrology and due to psychogenic or mild organic etiology, but the Sexology Department, Faculty of Medicine, Cairo Uni- 8 versity, Cairo 11562, Egypt. outcome of its use was, however, disappointing. E-mail: [email protected] Since the advent of sildenafil, there has been a Received 27 December 2006; revised 28 September 2007; resurgence of interest in ED and an increase in 9 accepted 25 October 2007; published online 17 April 2008 patients presenting with this disease. However, Combined effect of yohimbine and sildenafil AM Senbel and T Mostafa 410 several studies showed that administering sildenafil computed at the maximal effect after CN stimulation could not result in rigidity sufficient for satisfactory and SAP is the systemic arterial pressure computed sexual intercourse in 11–22% of patients with at the same time) was calculated. ICP/SAP 1 and psychogenic and 36–65% with organic ED.10,11 5 min after intravenous (i.v.) administration of the Combination drug therapy seems appealing to treat drug were compared to control cavernous responses patients with ED in whom monotherapy with in the same rat. Percentage potentiation in ICP sildenafil failed. Therefore, combining the centrally induced by the combinations was then statistically acting drug yohimbine with the peripheral condi- compared to that induced by every drug alone in the tioner sildenafil might be an attractive approach. previous set of experiments at the same time This study therefore was designed to investigate the interval. In this set of experiments, the drug(s) and effect of yohimbine on sildenafil-induced facilita- drug combinations were administered i.v.; the effect tion of centrally and peripherally mediated erectile was recorded 1 and 5 min after injection. A catheter process in male rats. was inserted into the femoral vein and used for i.v. drug administration. A time-matched control using saline vehicle was performed alongside with the Materials and methods original experiment.

Animals Experiments were carried out on male albino rats Intravascular cannulation and measurement of 200–350 g weight (approximately aged 4–6 months) blood pressure and female albino rats 150–200 g. All animals used Measurement of blood pressure and heart rate in rats were bred in the animal house at the Faculty of was made as described previously by Tseng et al.14 Pharmacy according to NIH guidelines, housed About 1 cm incision was made in the skin of the under constant environmental conditions, fed diets groin and the underlying muscles were cut, then the consisting of wheat or bread soaked in milk. Water femoral artery and vein were exposed and freed was allowed ad libitum. from underlying muscles. By a fine scissors or a 901 curved needle, a small incision was made in the artery near a closed tie through which a polyethylene catheter, 20 cm length filled with heparinized Measurement of ICP in rats saline, was introduced. The arterial catheter Experiments have been conducted by measuring was connected to the pressure transducer and intracavernosal pressure (ICP) changes elicited arterial blood pressure was displayed on a Grass by electrical stimulation of the cavernosal polygraph. nerve (CN) in anesthetized rats according to the method described by Giuliani et al.12 Male rats were anesthetized by intraperitoneal injection of thiopen- tal (50 mg kgÀ1), then the skin overlying the penis Behavioral testing in rats was incised, and the prepuce was degloved to Copulatory behavioral tests on rats were performed expose the corpora cavernosa. A 26-gauge needle as described previously by Butcher et al.15 Male and filled with heparinized saline was carefully inserted female rats were housed at room temperature for 1 into the corpus cavernosum on one side to measure week before the start of the experimental period, ICP. The needle was sealed to a polyethylene 50 three or four to a cage. All experiments were tubing of 20 cm length filled with heparinized performed between 1100 and 1500 hours in a sound saline, connected to a Gould-Statham pressure attenuated room.16 Sexual activity of rats was transducer (Oxnard, CA, USA). ICP was displayed evaluated after four selection mating tests with on a Grass polygraph (Model 7D, Grass Inst. Co., receptive females. After selection tests, animals MA, USA). were admitted to the experimental session if they Through a lower suprapubic midline incision, the had exhibited at least one ejaculation per test.17 The lateral prostate was dissected and the major pelvic normal coital pattern in male rats is characterized by ganglion was identified. The CN was unilaterally the following behavioral sequence: (1) mounting freed from its facial attachments and stimulated of the female followed by a penile intromission electrically using a bipolar platinum electrode lasting approximately 1/3 s, (2) withdrawal of the placed 3–4 mm distal to the major pelvic ganglion.13 penis and dismount and (3) a brief rest interval of The two poles of the electrode were separated by about 40–60 s. This sequence is then repeated 2 mm and the electrode was connected to an several times until ejaculation occurs with the electronic stimulator (Letica, Panlab, Model 12106/ terminal intromission of the series. Ejaculation was 150, Spain). CN was stimulated for 1 min, the pulse identified by vigorous thrush and longer intro- parameters were 5 V, 1 ms duration and 0.5–10 Hz mission period, and was always followed by a frequency. Maximum rise in ICP during nerve resting period (post-ejaculatory interval (PEI)) of stimulation was measured. ICP/SAP ratio (ICP is 10–15 min.15

International Journal of Impotence Research Combined effect of yohimbine and sildenafil AM Senbel and T Mostafa 411 Estrous was induced in the stimulus female by level. Throughout the manuscript ‘n’ indicates the means of a combined treatment of estrogen and number of rats. progesterone. Forty-eight hours before testing, the females were subcutaneously injected with À1 À1 60 mgkg estradiol benzoate followed by 1 mg kg Results progesterone 4 h prior the observation period. After a 10-min adaptation period in the observation cage, Effect of sildenafil on nerve-stimulated erectile a receptive female was presented to the male by responses dropping it gently into the cage. By direct observa- This set of experiments began with 3–5 CN stimulation, the following behavioral parameters were tions at different frequencies to select a suitable recorded: frequency that produced a submaximal increase in (1) mount latency (ML): the time from introduction ICP. The submaximal frequency (usually lying of the female to the occurrence of the first mount; between 1 and 6 Hz) was then repeated twice; a (2) intromission latency (IL): the time from intro- reproducible response was to be obtained. The drug duction of the female to the occurrence of the under test was then injected i.v. and the cavernous first intromission; responses to CN stimulation were recorded for 5 min (3) ejaculation latency (EL): time from the first after drug administration (Figure 1). Sildenafil intromission to ejaculation; produced potentiation of neurogenic erectile res- (4) PEI: time from ejaculation to the subsequent ponse at all concentrations used with significant À1 intromission; levels attained at concentrations X10 mmol kg . (5) intromission frequency (IF): number of intromis- Electrical stimulation of the CN produced an ICP/ sions preceding ejaculation; SAP value of 0.413±0.075 mm Hg in the presence of À1 (6) ejaculation frequency (EF): number of ejacula- 1 mmol kg sildenafil compared with 0.095± tion in a session; and 0.016 mm Hg in its absence 1 min after i.v. injection, (7) intercopulatory interval (ICI): average interval n ¼ 8 (Figure 2). between successive intromissions (EL/IF).

Testing sessions lasted 30 min. Each male animal Effect of yohimbine on nerve-stimulated erectile served as its own control. Each rat was used responses only twice, one time as a control and the second Yohimbine (0.1 mmol kgÀ1 to 1 mmol kgÀ1) failed to (after at least 48 h) as treated. The drug, combination increase ICP/SAP significantly compared to control or vehicle (saline) was injected intraperitoneally values, neither at 1 min nor at 5 min after i.v. 1 h before the testing session. The time between injection, n ¼ 7 (Figure 3). the two periods (control and treated) should be at least 48 h. Effect of sildenafil/yohimbine combinations on Statistics nerve-stimulated erectile responses Values were expressed as mean±s.e.m. Student’s ICP rise in response to electrical stimulation of the t-test was used for the analysis of paired data. For CN was recorded before and after the i.v. injection of multiple comparison, one-way analysis of variance combinations of yohimbine 1 mmol kg-1 with increas- (ANOVA or F-test) followed by Dunnet or Student– ing doses of sildenafil (1 mmol kgÀ1 to 1 mmol kgÀ1). Newman–Keuls post-test was performed. The criter- The effect of all combinations tested was signifi- ion for statistical significance was set at the 0.05 cantly greater than the effect of yohimbine alone.

Figure 1 Representative tracing showing the effect of intravenous administration of sildenafil on changes of intracavernosal pressure and mean systemic arterial pressure associated with cavernous nerve-stimulated penile erection in anesthetized rats.

International Journal of Impotence Research Combined effect of yohimbine and sildenafil AM Senbel and T Mostafa 412

Figure 2 Effect of i.v. sildenafil (1 mmol kgÀ1 to 1 mmol kgÀ1) on erectile responses to electrical stimulation of the cavernous nerve, 1 min (a) and 5 min (b) after injection. Results are expressed as mean±s.e.m. of eight experiments. *Denotes significant difference compared to control at the level of Po0.05. ICP, intracavernosal pressure; i.v., intravenous; SAP, systemic arterial pressure.

Figure 3 Effect of i.v. yohimbine (0.1 mmol kgÀ1 to 1 mmol kgÀ1) on erectile responses to electrical stimulation of the cavernous nerve, 1 min (a) and 5 min (b) after injection. Results are expressed as mean±s.e.m. of seven experiments. ICP, intracavernosal pressure; i.v., intravenous; SAP, systemic arterial pressure.

One minute after i.v. administration, percentage their combination was calculated and statistically potentiation of ICP/SAP induced by a combination compared to that of sildenafil alone, yohimbine alone of 10 mmol kgÀ1 sildenafil and 1 mmol kgÀ1 yohim- and that of the algebraic sum of their individual bine was significantly greater than the effect of effects. The effect of combination (1 mmol kgÀ1 sildenafil alone and that of the algebraic sum of yohimbine þ 10 mmol kgÀ1 sildenafil, n ¼ 8) on intro- individual effects, indicating a synergistic action. mission frequency and ICI was significantly different Similar action was observed with a higher dose from the effect of yohimbine alone. Combining low combination (1 mmol kgÀ1 sildenafil and 1 mmol kgÀ1 doses of sildenafil and yohimbine added a further yohimbine) 5 min after injection, n ¼ 7 (Figure 4). advantage: a potentiation of the number of erections/ session compared to none for either sildenafil or yohimbine alone (Figures 5–7).

Effect of sildenafil, yohimbine and sildenafil/ yohimbine combination on mating behavioral parameters Effect on SAP Intraperitoneal injection of yohimbine (1 mmol kgÀ1), For each i.v. injection, the SAP (mm Hg) was n ¼ 8 in male rats increased sexual motivation measured directly at the time of injection as well parameters. It significantly reduced ML, EL, PEI as 1 and 5 min after administration of the drug(s). and ICI compared to control values. Sildenafil Values were then compared to basal blood pressure (10 mmol kgÀ1, n ¼ 8) significantly reduced time to values just before injection. The reduction in SAP the first intromission and ejaculation. Sildenafil induced by sildenafil was more pronounced than showed a facilitator effect on erection with signifi- that induced by yohimbine, especially at high doses. cant reduction in intromissions required to achieve Combinations of drugs tested did not produce a ejaculation. Percent change (potentiation or inhibi- further decrease in SAP, compared to the additive tion) of each of the mating parameters induced by effect of sildenafil and yohimbine (Table 1).

International Journal of Impotence Research Combined effect of yohimbine and sildenafil AM Senbel and T Mostafa 413

Figure 4 Effect of sildenafil (1 mmol kgÀ1 to 1 mmol kgÀ1), yohimbine (1 mmol kgÀ1) and their combination on erectile responses to electrical stimulation of the cavernous nerve, 1 min (a) and 5 min (b) after injection. Values are expressed as mean±s.e.m. of seven experiments. $Denotes significant difference compared to sildenafil group at the level of Po0.05, #denotes significant difference compared to yohimbine group at the level of Po0.05 and *denotes significant difference compared to the sum of individual effects at the level of Po0.05. ICP, intracavernosal pressure; SAP, systemic arterial pressure.

Figure 5 Effect of yohimbine (1 mmol kgÀ1) on different copulatory parameters in male rats. Values are expressed as mean±s.e.m. of eight experiments. Rats were injected intraperitoneally with the drug 1 h before the 30 min experimental session. *Denotes significant difference compared to control at the level of Po0.05. Discussion tized rats.20 When sildenafil was tested alone, it succeeded to significantly reduce IL indicating a In the in vivo rat model where erection is induced by moderate potentiatory effect on sexual motivation, electrical stimulation of the CN, i.v. administration although the other sexual motivation-driven para- of sildenafil (10 mmol kgÀ1 to 1 mmol kgÀ1) dose meters (ML, PEI and ICI) were unaltered. EL and dependently increased ICP significantly compared number of intromissions preceding ejaculation were to the control values. The current results are significantly reduced by the action of sildenafil in in agreement with previous reports, sildenafil rats, indicating a beneficial effect on copulatory was demonstrated to facilitate neurogenic penile performance, may be next to the peripheral facili- erections in rabbits18 and dogs,19 and are also in line tatory effect of sildenafil on erection. However, with the previously described results in anesthe- when tested in combination with yohimbine, both

International Journal of Impotence Research Combined effect of yohimbine and sildenafil AM Senbel and T Mostafa 414

Figure 6 Effect of sildenafil (10 mmol kgÀ1) on different copulatory parameters in male rats. Values are expressed as mean±s.e.m. of eight experiments. Rats were injected intraperitoneally with the drug 1 h before the 30 min experimental session. *Denotes significant difference compared to control at the level of Po0.05.

decreased the intromission frequency and increased number of ejaculations per session indicating an increase in copulatory performance and potency. The synergistic effect of this combination on sexual and copulatory behaviors may be attributed to the combination of the peripheral effects of sildenafil on erectile function with the central effect of yohimbine and sildenafil, to a lesser extent, on male sexual behaviors. Giuliani et al.21 reported that sildenafil acts not only peripherally but also centrally since oral administration of sildenafil (1 mg kgÀ1) in rats modified both sexual and ejaculatory mechanisms of copulation. However, it was demonstrated that sildenafil did not improve sexual function in men

À1 without ED and it did not induce erections in young Figure 7 Comparison of the effect of sildenafil (10 mmol kg ), 22 yohimbine (1 mmol kgÀ1) and sildenafil/yohimbine combination healthy men. The moderate results of sildenafil on on mount latency (ML), intromission latency (IL), ejaculation mating parameters, as demonstrated in the present latency (EL), post-ejaculatory interval (PEI), intromission fre- study, are consistent with the low expression of quency (IF), ejaculation frequency (EF) and intercopulatory PDE5 in the brain, as the most widely expressed PDE ± interval (ICI) in male rats. Values are expressed as mean s.e.m. isozymes in the brain are PDE1 and 2, which are of eight experiments. Rats were injected intraperitoneally with 23 the combination 1 h before the 30 min experimental session. related to memory. Some investigators reported $Denotes significant difference compared to the sildenafil group that sildenafil enhanced object recognition memory at the level of Po0.05, #denotes significant difference compared * and that vardenafil increased cGMP concentrations to the yohimbine group at the level of Po0.05 and denotes in neural fibers of the hippocampal region in rats.24 significant difference compared to the sum of individual effects at the level of Po0.05. As for yohimbine, it failed to peripherally modu- late erectile functions; it did not potentiate neurogenic erections after systemic administration injected intraperitoneally, it reduced mount, intro- in rats. Previous studies demonstrated that yohim- mission and ELs and ICI, indicating an increase in bine potently relaxed phenylephrine precontracted arousal and motivation. The combination potently strips of rabbit corpus cavernosum.25 Consequently,

International Journal of Impotence Research Combined effect of yohimbine and sildenafil AM Senbel and T Mostafa 415 Table 1 Effect of sildenafil, yohimbine and sildenafil/yohimbine combination on SAP

Treatment Decrease in SAP (mm Hg)

At time of injection 1 min after injection 5 min after injection

Sildenafil 10 mmol kgÀ1 (8) 6.333±0.718 0.916±0.49* (P ¼ 0.036) 1.08±0.042 Yohimbine 1 mmol kgÀ1 (7) 3.50±1.345 1.25±0.496 1.25±0.283 Combination (7) 4.40±0.624 2.80±0.654 1.40±0.906 Sildenafil 1 mmol kgÀ1 (8) 27.50±1.058* (Po0.0001) 4.67±0.452* (P ¼ 0.0059) 1.75±0.074* (P ¼ 0.0162) Yohimbine 1 mmol kgÀ1 (7) 3.50±1.345* (Po0.0001) 1.25±0.496* (P ¼ 0.0219) 1.25±0.283* (P ¼ 0.0082) Combination (7) 16.8±0.937 2.80±0.318 2.80±0.40

Abbreviation: SAP, systemic arterial pressure. Values are expressed as mean±s.e.m. Values in parentheses in column 1 indicate number of experiments. *Indicates statistical difference in comparison to the combination group. it could be expected that yohimbine would induce These results are in line with previous studies in erection in absence of sexual stimulation; however, animals showing that the drug has a remarkable contrary results were obtained in this study. This positive effect on sexual performance.29–31 It is effect has not been reported in vivo in animals or in reported that yohimbine facilitates sexual arousal humans. Yohimbine had no effect on erectility when by acting on a2-adrenergic receptors in central given intracavernosally in humans.26 However, nervous system.2,32,33 Interestingly, yohimbine may yohimbine, in a few number of combinations tested stimulate serotonin receptors in the brain,32 and in the current study, was able to enhance the effect enhanced serotoninergic transmission is reported to of sildenafil synergistically on erectile responses stimulate oxytocin secretion in the blood of male induced by electrical stimulation of the CN. In rats, which in turn enhances sexual behavior and combination with yohimbine, the potentiatory effect erection.34 Furthermore, it was suggested that the of a higher dose of sildenafil was prolonged. Maggi dopaminergic system might be the final pathway for 2 35 et al. indicated that yohimbine is a mixed a1- and yohimbine-induced behavior expression. a2-adrenergic receptor antagonist. It seems from the At all doses tested, sildenafil decreased blood latter study that a1-receptors antagonism predomi- pressure directly after injection in agreement with nates at high doses since yohimbine (1 Â 10À8 and the clinical studies of sildenafil in humans. After 1 Â 10À7 M) enhanced electrically induced contrac- single therapeutic doses, there was a non-dose- tions in isolated corpus cavernosum while dependent mild and transient decrease in blood 1 Â 10À6 M inhibited them.27 The possible periph- pressure not associated with a significant effect on eral events following yohimbine administration heart rate.36 In another clinical trial, sildenafil was have been reported to be as follows: (1) inhibition reported to have a modest effect on blood pressure of the binding to post-junctional a2-receptors attenu- in normal subjects, producing an average decrease of ating contraction mediated by norepinephrine (NE); about 10 mm Hg after a single oral dose of 100 mg.37 (2) inhibition of pre-junctional a2-receptors on the Yohimbine-induced hypotension was short in adrenergic nerves resulting in an increase in the duration in low doses but long-lasting with higher release of NE and (3) inhibition of the binding to ones. Consistently, Lang et al.38 reported that i.v. pre-junctional a2-receptors in the nonadrenergic administration of yohimbine in rats caused a noncholinergic nerves decreasing the release of decrease in blood pressure and an increase in heart NO.28 The two latter mechanisms could be expected rate. Similar results were reported in cats.39 Clinical to be the cause behind the modest effect of studies revealed controversial results. One study yohimbine observed on ICP. On the other hand, the reported that yohimbine increased blood pressure in effect of some high doses of sildenafil on ICP volunteers by 15–20 mm Hg,40 while others showed increases in response to electrical stimulation was that it did not significantly affect heart rate inhibited by combination with yohimbine. No data and blood pressure in normotensive subjects.41 It in literature about the mechanism of action of seems that the effect of yohimbine on cardiovascular sildenafil could explain this ‘negative synergism’, functions in human are minimal in relation to which remains a limitation of this study. Further those observed in rats. One of the advantages studies are needed to elaborate and explain this of the suggested sildenafil/yohimbine combination observation. is that no excessive hypotension was induced The effect of yohimbine on erectile function after i.v. administration. The effect of all combina- seems to be mostly centrally mediated, since in the tions tested on systemic blood pressure was present study, its acute intraperitoneal injection always lower than the algebraic sum of the reduced mount and ejaculation latencies, ICI and corresponding doses of sildenafil and yohimbine PEI, reflecting an increase in sexual motivation. when given alone.

International Journal of Impotence Research Combined effect of yohimbine and sildenafil AM Senbel and T Mostafa 416 In a similar attempt to study the effect of fibers on the surface of the prostate. Urology 1996; 47: combining yohimbine with a NO conditioner, Lebret 146–151. et al.42 demonstrated that on-demand administra- 14 Tseng CJ, Liu HY, Lin HC, Ger LP, Tung CS, Yen MH. Cardiovascular effects of nitric oxide in the brain stem nuclei tion of 3.25 g of the NO precursor L-arginine and of rats. Hypertension 1996; 27: 36–42. 6 mg yohimbine, administered 1–2 h before intended 15 Butcher LL, Butcher SG, Larsson K. Effects of apomorphine, sexual intercourse, significantly improved erectile amphetamnine, and nialiamide on tetrabenazine-induced function in patients with mild-to-moderate ED. The suppression of sexual behavior in the male rat. Eur J Pharmacol 1969; 7: 283–288. results of the current study, which still have to be 16 Drago F, Busa L, Benelli A, Bertilini A. 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