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Effect of Botulinum-A Toxin Injection into Bulbospongiosus Muscle on Ejaculation Latency in Male Rats Ege C. Serefoglu, MD, FECSM,*† Wayne R. Hawley, PhD,‡ George F. Lasker, MS,§ Elin M. Grissom, PhD,‡ Sree H. Mandava, MD,* Suresh C. Sikka, PhD,* Gary P. Dohanich, PhD,‡ and Wayne J.G. Hellstrom, MD, FACS* *Department of Urology, Tulane University School of Medicine, New Orleans, LA, USA; †Department of Urology, Bagcilar Training & Research Hospital, Istanbul, Turkey; ‡Department of Psychology, Tulane University, New Orleans, LA, USA; § Department of Pharmacology, Tulane University School of Medicine, New Orleans, LA, USA DOI: 10.1111/jsm.12553
ABSTRACT
Introduction. Premature ejaculation (PE) is the most common male sexual dysfunction. A variety of pharmacotherapeutic strategies have been employed to treat men suffering with lifelong PE. However, there are currently no pharmaceuticals approved by the U.S. Food and Drug Administration specifically designed for PE treatment. Aim. Given that the bulbospongiosus muscle is involved in the ejaculatory reflex in both humans and rodents and that local administration of botulinum-A can abolish muscle contractions, the current study examined the effect of injection of botulinum-A toxin into the bulbospongiosus muscle on the ejaculatory latency of male rats. Methods. After screening for normal sexual activity with sexually receptive female rats, 33 sexually experienced male Long-Evans rats (Harlan Laboratories, Indianapolis, IN, USA) underwent an additional four pretreatment sexual exposures over the course of the following week, during which all components of sexual behavior were video recorded by trained observers. On the day after their fourth experience, rats were anesthetized and received a single injection of either 0.5 unit (n = 11) or 1 unit (n = 11) of botulinum-A toxin or saline vehicle (n = 11). Botulinum-A toxin was dissolved in 0.1 mL of saline vehicle and injected bilaterally into the bulbospongiosus muscle by the percutaneous route. Beginning 2 days after treatment, sexual behaviors were reexamined over the course of the following week on four separate occasions. Main Outcome Measures. The latency to achieve ejaculation, and the frequencies and latencies of mounts and intromissions were video recorded by trained observers in a blinded fashion. Results. Relative to pretreatment measurements, bilateral injection of saline vehicle into the bulbospongiosus muscle did not affect ejaculation latencies. However, rats treated with either 0.5 or 1 unit of botulinum-A toxin exhibited significantly longer latencies to achieve ejaculation relative to pretreatment performance. Of note, botulinum-A toxin did not affect the ability to achieve mounts, intromissions, or ejaculation. Conclusions. These results demonstrate that botulinum-A toxin injection into the bulbospongiosus muscle is a safe and effective treatment that extends ejaculatory latency in rats without affecting the ability to engage in sexual activity or achieve ejaculation. Further studies are required to evaluate this therapeutic concept in PE patients. Serefoglu EC, Hawley WR, Lasker GF, Grissom EM, Mandava SH, Sikka SC, Dohanich GP, and Hellstrom WJG. Effect of botulinum-A toxin injection into bulbospongiosus muscle on ejaculation latency in male rats. J Sex Med **;**:**–**. Key Words. Animal Models for Ejaculatory Behavior; Botulinum-A Toxin; Bulbospongiosus Muscle; Intromission; Premature Ejaculation; Sexual Behavior
© 2014 International Society for Sexual Medicine
J Sex Med **;**:**–**
2 Introduction
L
ifelong premature ejaculation (PE) is defined as “a male sexual dysfunction characterized by ejaculation that always or nearly always occurs prior to or within about one minute of vaginal penetration; the inability to delay ejaculation on all or nearly all vaginal penetrations; and negative personal consequences such as distress, bother, frustration, and/or the avoidance of sexual intimacy” [1]. This condition is believed to have a neurobiological origin. Thus, pharmacotherapy should in theory be the treatment of choice for patients with lifelong PE [2,3]. Although the prevalence of lifelong PE is only between 2.3% and 3.2% in the general population [4,5], these patients comprise the majority of men seeking treatment [6,7]. Unfortunately, there are currently no pharmaceuticals approved by the U.S. Food and Drug Administration specifically designed for PE treatment, whereas a short-acting selective serotonin reuptake inhibitor (SSRI) dapoxetine, has been approved for treatment of PE in more than 50 countries worldwide. Ejaculation is a spinal cord reflex, which is constituted by emission and expulsion phases [8]. During expulsion, rhythmic contractions of the bulbospongiosus and ischiocavernosus muscles propel semen antegrade through the bulbar and penile urethra. Botulinum-A toxin is a selective blocker of acetylcholine release from nerve endings and inhibits neural transmission when injected into muscle [9]. Safe and effective urological indications for botulinum-A toxin include neurogenic detrusor over activity [10], detrusorsphincter dyssynergia [11], motor and sensory urge syndrome [12], and more recently, chronic prostatic pain [13]. The concept of inhibiting stereotyped rhythmic contractions of the bulbospongiosus muscle with botulinum-A toxin injection for the treatment of lifelong PE was initially suggested in 2010 [14]. The purpose of this study was to examine the effect of botulinum-A toxin injection into the bulbospongiosus muscle on the ejaculatory latency of normal sexually active male rats.
Materials and Methods
The Institutional Animal Care and Use Committee, in accordance with the National Institutes of Health policies guide for the care and use of laboratory animals, approved the experiments and procedures performed. J Sex Med **;**:**–**
Serefoglu et al.
Study Animals Long-Evans male and female rats (Harlan Laboratories, Indianapolis, IN, USA) received at 65 days of age were used for this study. Experimental males were housed singly to prevent aggressive encounters with cage mates, whereas stimulus females were housed in groups of three per cage. Rats were maintained on reversed light–dark cycle (12:12, lights off at 07:00 pm) with food and water available ad libitum. Ovariectomy of Stimulus Females Female rats, used as stimuli in sexual behavior tests, were ovariectomized aseptically via bilateral incisions under anesthesia induced by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (7 mg/kg). Following excision of each ovary, blood vessels were ligated, the muscle wall incisions were closed with sterile suture, and the skin incisions were closed with sterile wound clips. Females received free access to ibuprofen in drinking water to reduce postsurgical pain and were monitored daily throughout recovery. On the day of each sexual behavior test, ovariectomized rats were brought into sexual receptivity by intramuscular injections of estradiol benzoate (10 μg) at 48 and 24 hours before testing and progesterone (500 μg) at 5 hours before testing. Both steroids were delivered in 0.1 mL of sesame oil. Sexual Behavior Over the course of 1 week, male rats were screened for mounting behavior on four separate occasions. Males were tested in one of four equal-sized compartments within a 75-gallon glass aquaria (46 cm wide × 122 cm long × 53 cm high) lined with woodchip bedding in the presence of a sexually receptive female. Experienced observers recorded the latencies and frequencies of mounts, intromissions, and ejaculations to assess sexual function. The mount latency (time from presentation of the female to the first mount/intromission), intromission latency (time from presentation of the female to the first intromission), ejaculation latency (time from the first intromission to an ejaculation), and postejaculation interval (time from an ejaculation to the next intromission) were recorded. This paradigm has been previously employed to study the psychopharmacology of male rat ejaculatory behavior [8,15,16] and to assess the pharmacological effects of several potential agents for PE treatment [17,18]. Each behavior test was terminated following the postejaculation interval or after 30
3
Botulinum-A Toxin Injection for PE
significance level was set as P < 0.05. Analysis was performed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). Results
Figure 1 Percutaneous injection sites of 0.5 and 1 unit of botulinum-A toxin and the saline vehicle
minutes had elapsed without ejaculation. Following the initial screening for mounting behavior, sexually active males (n = 33) received an additional four sexual experiences over the course of the following week, which was just prior to botulinum-A toxin treatment.
Injection of Botulinum-A Toxin After completing the baseline behavior tests, male rats were randomly divided equally into three groups to receive a single injection of either 0.5 (n = 11) or 1 unit (n = 11) of botulinum-A toxin Botox 50 (Allergan Inc., Irvine, CA, USA) or saline vehicle (n = 11). Rats were first anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (7 mg/kg). The botulinum-A toxin was delivered bilaterally in a 0.1 mL of saline vehicle and injected percutaneously into the bulbospongiosus muscle (Figure 1). As bulbospongiousus muscles were prominent with palpation in anesthetized rats, targeting these muscles for injection was not difficult (Figure 2). Beginning 2 days after treatment, sexual behaviors were reexamined over the course of the following week on four separate occasions. Data Analysis Geometric means of all parameters evaluated were calculated, and the Wilcoxon test was used to compare sexual performance before and after botulinum-A toxin injection. Moreover, arithmetic means of ejaculation latency time during each behavioral experiment were assessed for treatment groups separately, and the outcomes were compared with two-factor repeated measures analysis of variance and Kruskall–Wallis test. Statistical
Pretreatment ejaculation latencies (P = 0.330), mounting frequencies (P = 0.227), mounting latencies (P = 0.884), intromission frequencies (P = 0.06), and intromission latencies (P = 0.241) were similar among three groups (Table 1). Bilateral injection of saline into the bulbospongiosus muscle did not impact ejaculation latencies (P = 0.248). However, rats treated with either 0.5 or 1 unit botulinum-A toxin exhibited significantly longer ejaculatory latencies compared with their pretreatment performance (0.5 unit: 314.6 ± 193.1 to 507.6 ± 277.8 seconds, P = 0.021; 1 unit: 264.4 ± 129.1 to 598.2 ± 352.5 seconds, P = 0.008; Table 1). Although botulinum-A toxin injection increased ejaculatory latencies relative to preinjection tests, a Kruskal–Wallis test could not demonstrate a statistical significance difference between the three groups across the postinjection tests (P = 0.438). Mean ejaculation latency reached its peak on the third post-treatment test (11 days after injection) and decreased sharply on the fourth post-treatment test (14 days after injection) at both doses (Figure 3). Botulinum-A toxin did not affect the ability to mount or achieve intromissions. However, botulinum-A toxin altered some parameters of sexual behavior including decreasing the frequency of mounts at both of the low (P = 0.05) and
Figure 2 Demonstration of bulbospongiosus muscles (arrows) after the completion of experiments. These muscles were prominent with palpation thus targeting them for injection was not difficult.
J Sex Med **;**:**–**
4 Table 1
Serefoglu et al. Geometric mean frequencies and latencies of mounts and intromissions, and latencies of ejaculations
Botulinum-A toxin dose Ejaculation latencies (seconds) None (saline) Low (0.5 unit) High (1.0 unit) Mount frequencies None (saline) Low (0.5 unit) High (1.0 unit) Mount latencies (seconds) None (saline) Low (0.5 unit) High (1.0 unit) Intromission frequencies None (saline) Low (0.5 unit) High (1.0 unit) Intromission latencies (seconds) None (saline) Low (0.5 unit) High (1.0 unit)
Pretreatment
Post-treatment
*P
347.5 (166.3) 314.6 (193.1) 264.4 (129.1)
401.7 (248.8) 507.6 (277.8) 598.2 (352.5)
0.248 *0.021 *0.008
14.4 (10.9) 12.7 (6.5) 23.8 (21.3)
11.5 (6.6) 7.3 (4.9) 8.1 (4.9)
0.790 *0.050 *0.033
7.1 (4.9) 5.9 (2.8) 8.1 (6.9)
15.6 (6.4) 22.3 (9.8) 12.9 (8.4)
*0.008 *0.003 0.182
10.8 (2.5) 11.0 (3.1) 14.6 (4.6)
14.1 (6.9) 12.5 (3.5) 11.3 (3.8)
0.374 0.248 0.155
15.2 (14.1) 11.6 (7.4) 7.7 (3.5)
10.0 (3.9) 8.3 (4.3) 12.3 (5.8)
0.374 0.093 *0.026
*Wilcoxon test Geometric mean (standard deviation) from four behavioral tests given prior to treatment and four behavioral tests given following treatment
high (P = 0.03) doses; increasing the latency of mounts at the low dose (P = 0.003); and increasing the latency of intromissions at the high dose (P = 0.026). Nevertheless, all rats treated with botulinum-A toxin displayed normal mating patterns and achieved ejaculation. Of note, one rat in the placebo group did not ejaculate on the third pretreatment test, whereas three (one rat in the low-dose and two in high-dose groups) and two (one rat in the placebo group and one in the highdose group) rats did not ejaculate on third and fourth post-treatment test. None of the rats died or had any observable health problems after the injections.
lidocaine creams were the first medical treatment proposed for treating PE [22], the side effects of local anesthetics (e.g., penile hypoesthesia, transvaginal absorption causing in vaginal numbness, and penile/vaginal dermatitis) and difficulties in product application hinder its practical use [23,24]. Daily SSRI therapy exhibits a number of side effects such as insomnia, fatigue, nausea, constipation, decreased libido, anorgasmia, erectile dysfunction, and male infertility [25–29]. Therefore, discontinuation rates for all off-label PE treatments are substantial [30]. The novel SSRI, dapoxetine, is the first oral agent designed for the treatment of PE. Unlike other SSRIs, it is quickly absorbed and
Discussion
J Sex Med **;**:**–**
1000 Mean latency (seconds)
The aim of this study was to evaluate the effects of botulinum-A toxin injection into the bulbospongiosus muscle on the ejaculatory latency of male rats. Our findings revealed that botulinum-A toxin injection into the bulbospongiosus muscle is a safe and effective modality to extend the ejaculatory latency in male rats without interfering with the ability to achieve intromission or ejaculation. Management of PE has evolved from traditional psychological and behavioral therapies to medicalized treatments [19]. Recent guidelines for PE treatment recommend the use of topical anesthetic creams and on-demand or daily SSRI therapy [20,21]. Although on-demand topical prilocaine–
Injection
800 High
600
Low None 400
200 1
2
3
4 5 6 Behavioural Test
7
8
Figure 3 Arithmetic mean of ejaculation latency observed during each behavioral test. Kruskall–Wallis test revealed that there was a significant difference at the second (P = 0.030), third (P = 0.028), and fourth (P = 0.019) posttreatment tests.
5
Botulinum-A Toxin Injection for PE rapidly cleared from the body [31] and proposed for on-demand use. In spite of numerous studies demonstrating the efficacy and safety of dapoxetine [31–34], global approval by regulatory bodies is incomplete. A recent study by Mondaini et al. [35] reported that 20% of PE patients have not used dapoxetine. Of the patients who initiated dapoxetine treatment, only 10.4% continued using it after 1 year. The limited use of this drug suggests the need for better treatment alternatives for men suffering with PE. Considering the aforementioned limitations of the currently available treatments for PE, botulinum-A toxin injection may be a promising alternate approach. The long duration of neural block observed with botulinum-A toxin injection may enable PE patients to participate in spontaneous sexual activity without the need for oral pills or applying creams prior to sexual activity. Botulinum-A toxin injections into bulbospongiosus muscle would not cause any systemic side effects, as witnessed with SSRI treatments or the local side effects for both partners reported with topical creams. A drawback of this treatment would be the need for periodic, mildly uncomfortable perineal injections. Inhibiting the contractions of the bulbospongiosus muscle with botulinum-A toxin may also cause temporary loss of ejaculation. Therefore, optimal dose titration in humans needs to be well established in future clinical trials. While the treatment of PE with botulinum-A toxin injections into the bulbospongiosus muscle was initially hypothesized in 2010 [14], Gaxiola et al. [36] have recently described other adaptation of this method for treating PE. These authors have proposed locally injecting 25 and 50 units of a botulinum-A toxin into the penis to prolong ejaculatory latency and thereby treat PE. In another patent application [37], these same authors have proposed a local administration of botulinum-A toxin into the frenulum, prepuce, or glans penis for treating PE. However, because of penile vascularity, it is likely that botulinum-A toxin injected into the penis could immediately diffuse into the systemic circulation resulting in widespread effects beyond the genitalia. Similarly, injecting botulinum-A toxin into the prepuce or frenulum of the penis will have little effect on ejaculatory latency because botulinum-A toxin does not exhibit any local anesthetic effect. Notably, the latter two proposals for a patent have not been based on a publication of such an experiment in a peer-reviewed journal yet.
The limitations of this study must also be addressed. The difference between post-treatment geometric mean ejaculatory latency among three groups could not reach a statistically significant level, probably due to the low sample size and high variability in ejaculation latency. Therefore, future studies must include higher number of animals. Moreover, histopathological examination of the bulbospongiosus muscle along with neighboring tissues could confirm the localized impact of botulinum-A toxin injection.
Conclusion
The current results from these rat studies demonstrate that botulinum-A toxin injection into the bulbospongiosus muscle is a safe and effective means to prolong ejaculatory latency without affecting other aspects of sexual behavior. Further studies in humans are required to evaluate and corroborate the therapeutic benefit of botulinum-A toxin injection into the bulbospongiosus muscle in the treatment of PE.
Acknowledgments
The authors would like to thank Theodore R. Saitz, Mehmet Berktas, Christopher Frederick, Jessica M. Fawcett-Patel, Kelly S. Hodges, Harriet E Barratt, Sarah E. Marino, Sarah S. Bromley-Dulfano, Nicholas G. Stathopoulos, and Nicole M. Moody for their contributions. Corresponding Author: Ege Can Serefoglu, MD, FECSM, Department of Urology, Bagcilar Training & Research Hospital, Cinnah Caddesi No. 47, 06680 Cankaya-Ankara, Turkey. Tel: +90-312-440-0333; Fax: +90-312-438-2792; E-mail: egecanserefoglu @hotmail.com Conflict of Interest: Ege Can Serefoglu and Wayne J.G. Hellstrom are consultants for Allergan Inc., Irwine, CA, USA.
Statement of Authorship
Category 1 (a) Conception and Design Ege C. Serefoglu; Elin M. Grissom; Wayne R. Hawley; Gary P. Dohanich (b) Acquisition of Data Wayne R. Hawley; George F. Lasker; Suresh C. Sikka; Elin M. Grissom; Sree H. Mandava (c) Analysis and Interpretation of Data Ege C. Serefoglu; Wayne R. Hawley J Sex Med **;**:**–**
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Category 2 (a) Drafting the Article Ege C. Serefoglu; Wayne R. Hawley (b) Revising It for Intellectual Content Gary P. Dohanich; Wayne J.G. Hellstrom
Category 3 (a) Final Approval of the Completed Article Ege C. Serefoglu; Gary P. Dohanich References 1 McMahon CG, Althof SE, Waldinger MD, Porst H, Dean J, Sharlip ID, Adaikan PG, Becher E, Broderick GA, Buvat J, Dabees K, Giraldi A, Giuliano F, Hellstrom WJ, Incrocci L, Laan E, Meuleman E, Perelman MA, Rosen RC, Rowland DL, Segraves R. An evidence-based definition of lifelong premature ejaculation: Report of the International Society for Sexual Medicine (ISSM) ad hoc committee for the definition of premature ejaculation. J Sex Med 2008;5:1590–606. 2 Waldinger MD. Recent advances in the classification, neurobiology and treatment of premature ejaculation. Adv Psychosom Med 2008;29:50–69. 3 Waldinger MD, Schweitzer DH. Changing paradigms from a historical DSM-III and DSM-IV view toward an evidencebased definition of premature ejaculation. Part II—Proposals for DSM-V and ICD-11. J Sex Med 2006;3:693–705. 4 Serefoglu EC, Yaman O, Cayan S, Asci R, Orhan I, Usta MF, Ekmekcioglu O, Kendirci M, Semerci B, Kadioglu A. Prevalence of the complaint of ejaculating prematurely and the four premature ejaculation syndromes: Results from the Turkish Society of Andrology sexual health survey. J Sex Med 2011;8:540–8. 5 Gao J, Zhang X, Su P, Liu J, Xia L, Yang J, Shi K, Tang D, Hao Z, Zhou J, Liang C. Prevalence and factors associated with the complaint of premature ejaculation and the four premature ejaculation syndromes: A large observational study in China. J Sex Med 2013;10:1874–81. 6 Zhang X, Gao J, Liu J, Xia L, Yang J, Hao Z, Zhou J, Liang C. Distribution and factors associated with four premature ejaculation syndromes in outpatients complaining of ejaculating prematurely. J Sex Med 2013;10:1603–11. 7 Serefoglu EC, Cimen HI, Atmaca AF, Balbay MD. The distribution of patients who seek treatment for the complaint of ejaculating prematurely according to the four premature ejaculation syndromes. J Sex Med 2010;7(2 Pt 1):810–5. 8 Giuliano F, Clement P. Physiology of ejaculation: Emphasis on serotonergic control. Eur Urol 2005;48:408–17. 9 Whelchel DD, Brehmer TM, Brooks PM, Darragh N, Coffield JA. Molecular targets of botulinum toxin at the mammalian neuromuscular junction. Mov Disord 2004;19(suppl 8):S7–16. 10 Linsenmeyer TA. Use of botulinum toxin in individuals with neurogenic detrusor overactivity: State of the art review. J Spinal Cord Med 2013;36:402–19. 11 Chen CY, Liao CH, Kuo HC. Therapeutic effects of detrusor botulinum toxin A injection on neurogenic detrusor overactivity in patients with different levels of spinal cord injury and types of detrusor sphincter dyssynergia. Spinal Cord 2011;49:659–64. 12 Mangera A, Andersson KE, Apostolidis A, Chapple C, Dasgupta P, Giannantoni A, Gravas S, Madersbacher S. Contemporary management of lower urinary tract disease with botulinum toxin A: A systematic review of botox (onabotulinumtoxinA) and dysport (abobotulinumtoxinA). Eur Urol 2011;60:784–95.
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Botulinum-A Toxin Injection for PE 31 Pryor JL, Althof SE, Steidle C, Rosen RC, Hellstrom WJ, Shabsigh R, Miloslavsky M, Kell S. Efficacy and tolerability of dapoxetine in treatment of premature ejaculation: An integrated analysis of two double-blind, randomised controlled trials. Lancet 2006;368:929–37. 32 Porst H, McMahon CG, Althof SE, Sharlip I, Bull S, Aquilina JW, Tesfaye F, Rivas DA. Baseline characteristics and treatment outcomes for men with acquired or lifelong premature ejaculation with mild or no erectile dysfunction: integrated analyses of two phase 3 dapoxetine trials. J Sex Med 2010;7:2231–42. 33 Buvat J, Tesfaye F, Rothman M, Rivas DA, Giuliano F. Dapoxetine for the treatment of premature ejaculation: Results from a randomized, double-blind, placebocontrolled phase 3 trial in 22 countries. Eur Urol 2009;55: 957–67.
7 34 Kaufman JM, Rosen RC, Mudumbi RV, Tesfaye F, Hashmonay R, Rivas D. Treatment benefit of dapoxetine for premature ejaculation: Results from a placebo-controlled phase III trial. BJU Int 2009;103:651–8. 35 Mondaini N, Fusco F, Cai T, Benemei S, Mirone V, Bartoletti R. Dapoxetine treatment in patients with lifelong premature ejaculation: The reasons of a “waterloo”. Urology 2013;82: 620–4. 36 Gaxiola GM, Aguilar IE, Paz GP, inventors; Allergan, Inc. (Irvine, CA), assignee. Method for treating premature ejaculation with a botulinum neurotoxin. United States of America patent 8,329,193. 2012. 37 Gaxiola GM, Aguilar IE, Paz GP, inventors; Allergan, Inc. (Irvine, CA) assignee. Method for treating premature ejaculation with a botulinum neurotoxin. United States of America patent 8,147,848. 2012.
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Effect of Botulinum-A Toxin Injection into Bulbospongiosus Muscle on Ejaculation Latency in Male Rats Ege C. Serefoglu, MD, FECSM,*† Wayne R. Hawley, PhD,‡ George F. Lasker, MS,§ Elin M. Grissom, PhD,‡ Sree H. Mandava, MD,* Suresh C. Sikka, PhD,* Gary P. Dohanich, PhD,‡ and Wayne J.G. Hellstrom, MD, FACS* *Department of Urology, Tulane University School of Medicine, New Orleans, LA, USA; †Department of Urology, Bagcilar Training & Research Hospital, Istanbul, Turkey; ‡Department of Psychology, Tulane University, New Orleans, LA, USA; § Department of Pharmacology, Tulane University School of Medicine, New Orleans, LA, USA DOI: 10.1111/jsm.12553
ABSTRACT
Introduction. Premature ejaculation (PE) is the most common male sexual dysfunction. A variety of pharmacotherapeutic strategies have been employed to treat men suffering with lifelong PE. However, there are currently no pharmaceuticals approved by the U.S. Food and Drug Administration specifically designed for PE treatment. Aim. Given that the bulbospongiosus muscle is involved in the ejaculatory reflex in both humans and rodents and that local administration of botulinum-A can abolish muscle contractions, the current study examined the effect of injection of botulinum-A toxin into the bulbospongiosus muscle on the ejaculatory latency of male rats. Methods. After screening for normal sexual activity with sexually receptive female rats, 33 sexually experienced male Long-Evans rats (Harlan Laboratories, Indianapolis, IN, USA) underwent an additional four pretreatment sexual exposures over the course of the following week, during which all components of sexual behavior were video recorded by trained observers. On the day after their fourth experience, rats were anesthetized and received a single injection of either 0.5 unit (n = 11) or 1 unit (n = 11) of botulinum-A toxin or saline vehicle (n = 11). Botulinum-A toxin was dissolved in 0.1 mL of saline vehicle and injected bilaterally into the bulbospongiosus muscle by the percutaneous route. Beginning 2 days after treatment, sexual behaviors were reexamined over the course of the following week on four separate occasions. Main Outcome Measures. The latency to achieve ejaculation, and the frequencies and latencies of mounts and intromissions were video recorded by trained observers in a blinded fashion. Results. Relative to pretreatment measurements, bilateral injection of saline vehicle into the bulbospongiosus muscle did not affect ejaculation latencies. However, rats treated with either 0.5 or 1 unit of botulinum-A toxin exhibited significantly longer latencies to achieve ejaculation relative to pretreatment performance. Of note, botulinum-A toxin did not affect the ability to achieve mounts, intromissions, or ejaculation. Conclusions. These results demonstrate that botulinum-A toxin injection into the bulbospongiosus muscle is a safe and effective treatment that extends ejaculatory latency in rats without affecting the ability to engage in sexual activity or achieve ejaculation. Further studies are required to evaluate this therapeutic concept in PE patients. Serefoglu EC, Hawley WR, Lasker GF, Grissom EM, Mandava SH, Sikka SC, Dohanich GP, and Hellstrom WJG. Effect of botulinum-A toxin injection into bulbospongiosus muscle on ejaculation latency in male rats. J Sex Med **;**:**–**. Key Words. Animal Models for Ejaculatory Behavior; Botulinum-A Toxin; Bulbospongiosus Muscle; Intromission; Premature Ejaculation; Sexual Behavior
© 2014 International Society for Sexual Medicine
J Sex Med **;**:**–**
2 Introduction
L
ifelong premature ejaculation (PE) is defined as “a male sexual dysfunction characterized by ejaculation that always or nearly always occurs prior to or within about one minute of vaginal penetration; the inability to delay ejaculation on all or nearly all vaginal penetrations; and negative personal consequences such as distress, bother, frustration, and/or the avoidance of sexual intimacy” [1]. This condition is believed to have a neurobiological origin. Thus, pharmacotherapy should in theory be the treatment of choice for patients with lifelong PE [2,3]. Although the prevalence of lifelong PE is only between 2.3% and 3.2% in the general population [4,5], these patients comprise the majority of men seeking treatment [6,7]. Unfortunately, there are currently no pharmaceuticals approved by the U.S. Food and Drug Administration specifically designed for PE treatment, whereas a short-acting selective serotonin reuptake inhibitor (SSRI) dapoxetine, has been approved for treatment of PE in more than 50 countries worldwide. Ejaculation is a spinal cord reflex, which is constituted by emission and expulsion phases [8]. During expulsion, rhythmic contractions of the bulbospongiosus and ischiocavernosus muscles propel semen antegrade through the bulbar and penile urethra. Botulinum-A toxin is a selective blocker of acetylcholine release from nerve endings and inhibits neural transmission when injected into muscle [9]. Safe and effective urological indications for botulinum-A toxin include neurogenic detrusor over activity [10], detrusorsphincter dyssynergia [11], motor and sensory urge syndrome [12], and more recently, chronic prostatic pain [13]. The concept of inhibiting stereotyped rhythmic contractions of the bulbospongiosus muscle with botulinum-A toxin injection for the treatment of lifelong PE was initially suggested in 2010 [14]. The purpose of this study was to examine the effect of botulinum-A toxin injection into the bulbospongiosus muscle on the ejaculatory latency of normal sexually active male rats.
Materials and Methods
The Institutional Animal Care and Use Committee, in accordance with the National Institutes of Health policies guide for the care and use of laboratory animals, approved the experiments and procedures performed. J Sex Med **;**:**–**
Serefoglu et al.
Study Animals Long-Evans male and female rats (Harlan Laboratories, Indianapolis, IN, USA) received at 65 days of age were used for this study. Experimental males were housed singly to prevent aggressive encounters with cage mates, whereas stimulus females were housed in groups of three per cage. Rats were maintained on reversed light–dark cycle (12:12, lights off at 07:00 pm) with food and water available ad libitum. Ovariectomy of Stimulus Females Female rats, used as stimuli in sexual behavior tests, were ovariectomized aseptically via bilateral incisions under anesthesia induced by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (7 mg/kg). Following excision of each ovary, blood vessels were ligated, the muscle wall incisions were closed with sterile suture, and the skin incisions were closed with sterile wound clips. Females received free access to ibuprofen in drinking water to reduce postsurgical pain and were monitored daily throughout recovery. On the day of each sexual behavior test, ovariectomized rats were brought into sexual receptivity by intramuscular injections of estradiol benzoate (10 μg) at 48 and 24 hours before testing and progesterone (500 μg) at 5 hours before testing. Both steroids were delivered in 0.1 mL of sesame oil. Sexual Behavior Over the course of 1 week, male rats were screened for mounting behavior on four separate occasions. Males were tested in one of four equal-sized compartments within a 75-gallon glass aquaria (46 cm wide × 122 cm long × 53 cm high) lined with woodchip bedding in the presence of a sexually receptive female. Experienced observers recorded the latencies and frequencies of mounts, intromissions, and ejaculations to assess sexual function. The mount latency (time from presentation of the female to the first mount/intromission), intromission latency (time from presentation of the female to the first intromission), ejaculation latency (time from the first intromission to an ejaculation), and postejaculation interval (time from an ejaculation to the next intromission) were recorded. This paradigm has been previously employed to study the psychopharmacology of male rat ejaculatory behavior [8,15,16] and to assess the pharmacological effects of several potential agents for PE treatment [17,18]. Each behavior test was terminated following the postejaculation interval or after 30
3
Botulinum-A Toxin Injection for PE
significance level was set as P < 0.05. Analysis was performed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). Results
Figure 1 Percutaneous injection sites of 0.5 and 1 unit of botulinum-A toxin and the saline vehicle
minutes had elapsed without ejaculation. Following the initial screening for mounting behavior, sexually active males (n = 33) received an additional four sexual experiences over the course of the following week, which was just prior to botulinum-A toxin treatment.
Injection of Botulinum-A Toxin After completing the baseline behavior tests, male rats were randomly divided equally into three groups to receive a single injection of either 0.5 (n = 11) or 1 unit (n = 11) of botulinum-A toxin Botox 50 (Allergan Inc., Irvine, CA, USA) or saline vehicle (n = 11). Rats were first anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (7 mg/kg). The botulinum-A toxin was delivered bilaterally in a 0.1 mL of saline vehicle and injected percutaneously into the bulbospongiosus muscle (Figure 1). As bulbospongiousus muscles were prominent with palpation in anesthetized rats, targeting these muscles for injection was not difficult (Figure 2). Beginning 2 days after treatment, sexual behaviors were reexamined over the course of the following week on four separate occasions. Data Analysis Geometric means of all parameters evaluated were calculated, and the Wilcoxon test was used to compare sexual performance before and after botulinum-A toxin injection. Moreover, arithmetic means of ejaculation latency time during each behavioral experiment were assessed for treatment groups separately, and the outcomes were compared with two-factor repeated measures analysis of variance and Kruskall–Wallis test. Statistical
Pretreatment ejaculation latencies (P = 0.330), mounting frequencies (P = 0.227), mounting latencies (P = 0.884), intromission frequencies (P = 0.06), and intromission latencies (P = 0.241) were similar among three groups (Table 1). Bilateral injection of saline into the bulbospongiosus muscle did not impact ejaculation latencies (P = 0.248). However, rats treated with either 0.5 or 1 unit botulinum-A toxin exhibited significantly longer ejaculatory latencies compared with their pretreatment performance (0.5 unit: 314.6 ± 193.1 to 507.6 ± 277.8 seconds, P = 0.021; 1 unit: 264.4 ± 129.1 to 598.2 ± 352.5 seconds, P = 0.008; Table 1). Although botulinum-A toxin injection increased ejaculatory latencies relative to preinjection tests, a Kruskal–Wallis test could not demonstrate a statistical significance difference between the three groups across the postinjection tests (P = 0.438). Mean ejaculation latency reached its peak on the third post-treatment test (11 days after injection) and decreased sharply on the fourth post-treatment test (14 days after injection) at both doses (Figure 3). Botulinum-A toxin did not affect the ability to mount or achieve intromissions. However, botulinum-A toxin altered some parameters of sexual behavior including decreasing the frequency of mounts at both of the low (P = 0.05) and
Figure 2 Demonstration of bulbospongiosus muscles (arrows) after the completion of experiments. These muscles were prominent with palpation thus targeting them for injection was not difficult.
J Sex Med **;**:**–**
4 Table 1
Serefoglu et al. Geometric mean frequencies and latencies of mounts and intromissions, and latencies of ejaculations
Botulinum-A toxin dose Ejaculation latencies (seconds) None (saline) Low (0.5 unit) High (1.0 unit) Mount frequencies None (saline) Low (0.5 unit) High (1.0 unit) Mount latencies (seconds) None (saline) Low (0.5 unit) High (1.0 unit) Intromission frequencies None (saline) Low (0.5 unit) High (1.0 unit) Intromission latencies (seconds) None (saline) Low (0.5 unit) High (1.0 unit)
Pretreatment
Post-treatment
*P
347.5 (166.3) 314.6 (193.1) 264.4 (129.1)
401.7 (248.8) 507.6 (277.8) 598.2 (352.5)
0.248 *0.021 *0.008
14.4 (10.9) 12.7 (6.5) 23.8 (21.3)
11.5 (6.6) 7.3 (4.9) 8.1 (4.9)
0.790 *0.050 *0.033
7.1 (4.9) 5.9 (2.8) 8.1 (6.9)
15.6 (6.4) 22.3 (9.8) 12.9 (8.4)
*0.008 *0.003 0.182
10.8 (2.5) 11.0 (3.1) 14.6 (4.6)
14.1 (6.9) 12.5 (3.5) 11.3 (3.8)
0.374 0.248 0.155
15.2 (14.1) 11.6 (7.4) 7.7 (3.5)
10.0 (3.9) 8.3 (4.3) 12.3 (5.8)
0.374 0.093 *0.026
*Wilcoxon test Geometric mean (standard deviation) from four behavioral tests given prior to treatment and four behavioral tests given following treatment
high (P = 0.03) doses; increasing the latency of mounts at the low dose (P = 0.003); and increasing the latency of intromissions at the high dose (P = 0.026). Nevertheless, all rats treated with botulinum-A toxin displayed normal mating patterns and achieved ejaculation. Of note, one rat in the placebo group did not ejaculate on the third pretreatment test, whereas three (one rat in the low-dose and two in high-dose groups) and two (one rat in the placebo group and one in the highdose group) rats did not ejaculate on third and fourth post-treatment test. None of the rats died or had any observable health problems after the injections.
lidocaine creams were the first medical treatment proposed for treating PE [22], the side effects of local anesthetics (e.g., penile hypoesthesia, transvaginal absorption causing in vaginal numbness, and penile/vaginal dermatitis) and difficulties in product application hinder its practical use [23,24]. Daily SSRI therapy exhibits a number of side effects such as insomnia, fatigue, nausea, constipation, decreased libido, anorgasmia, erectile dysfunction, and male infertility [25–29]. Therefore, discontinuation rates for all off-label PE treatments are substantial [30]. The novel SSRI, dapoxetine, is the first oral agent designed for the treatment of PE. Unlike other SSRIs, it is quickly absorbed and
Discussion
J Sex Med **;**:**–**
1000 Mean latency (seconds)
The aim of this study was to evaluate the effects of botulinum-A toxin injection into the bulbospongiosus muscle on the ejaculatory latency of male rats. Our findings revealed that botulinum-A toxin injection into the bulbospongiosus muscle is a safe and effective modality to extend the ejaculatory latency in male rats without interfering with the ability to achieve intromission or ejaculation. Management of PE has evolved from traditional psychological and behavioral therapies to medicalized treatments [19]. Recent guidelines for PE treatment recommend the use of topical anesthetic creams and on-demand or daily SSRI therapy [20,21]. Although on-demand topical prilocaine–
Injection
800 High
600
Low None 400
200 1
2
3
4 5 6 Behavioural Test
7
8
Figure 3 Arithmetic mean of ejaculation latency observed during each behavioral test. Kruskall–Wallis test revealed that there was a significant difference at the second (P = 0.030), third (P = 0.028), and fourth (P = 0.019) posttreatment tests.
5
Botulinum-A Toxin Injection for PE rapidly cleared from the body [31] and proposed for on-demand use. In spite of numerous studies demonstrating the efficacy and safety of dapoxetine [31–34], global approval by regulatory bodies is incomplete. A recent study by Mondaini et al. [35] reported that 20% of PE patients have not used dapoxetine. Of the patients who initiated dapoxetine treatment, only 10.4% continued using it after 1 year. The limited use of this drug suggests the need for better treatment alternatives for men suffering with PE. Considering the aforementioned limitations of the currently available treatments for PE, botulinum-A toxin injection may be a promising alternate approach. The long duration of neural block observed with botulinum-A toxin injection may enable PE patients to participate in spontaneous sexual activity without the need for oral pills or applying creams prior to sexual activity. Botulinum-A toxin injections into bulbospongiosus muscle would not cause any systemic side effects, as witnessed with SSRI treatments or the local side effects for both partners reported with topical creams. A drawback of this treatment would be the need for periodic, mildly uncomfortable perineal injections. Inhibiting the contractions of the bulbospongiosus muscle with botulinum-A toxin may also cause temporary loss of ejaculation. Therefore, optimal dose titration in humans needs to be well established in future clinical trials. While the treatment of PE with botulinum-A toxin injections into the bulbospongiosus muscle was initially hypothesized in 2010 [14], Gaxiola et al. [36] have recently described other adaptation of this method for treating PE. These authors have proposed locally injecting 25 and 50 units of a botulinum-A toxin into the penis to prolong ejaculatory latency and thereby treat PE. In another patent application [37], these same authors have proposed a local administration of botulinum-A toxin into the frenulum, prepuce, or glans penis for treating PE. However, because of penile vascularity, it is likely that botulinum-A toxin injected into the penis could immediately diffuse into the systemic circulation resulting in widespread effects beyond the genitalia. Similarly, injecting botulinum-A toxin into the prepuce or frenulum of the penis will have little effect on ejaculatory latency because botulinum-A toxin does not exhibit any local anesthetic effect. Notably, the latter two proposals for a patent have not been based on a publication of such an experiment in a peer-reviewed journal yet.
The limitations of this study must also be addressed. The difference between post-treatment geometric mean ejaculatory latency among three groups could not reach a statistically significant level, probably due to the low sample size and high variability in ejaculation latency. Therefore, future studies must include higher number of animals. Moreover, histopathological examination of the bulbospongiosus muscle along with neighboring tissues could confirm the localized impact of botulinum-A toxin injection.
Conclusion
The current results from these rat studies demonstrate that botulinum-A toxin injection into the bulbospongiosus muscle is a safe and effective means to prolong ejaculatory latency without affecting other aspects of sexual behavior. Further studies in humans are required to evaluate and corroborate the therapeutic benefit of botulinum-A toxin injection into the bulbospongiosus muscle in the treatment of PE.
Acknowledgments
The authors would like to thank Theodore R. Saitz, Mehmet Berktas, Christopher Frederick, Jessica M. Fawcett-Patel, Kelly S. Hodges, Harriet E Barratt, Sarah E. Marino, Sarah S. Bromley-Dulfano, Nicholas G. Stathopoulos, and Nicole M. Moody for their contributions. Corresponding Author: Ege Can Serefoglu, MD, FECSM, Department of Urology, Bagcilar Training & Research Hospital, Cinnah Caddesi No. 47, 06680 Cankaya-Ankara, Turkey. Tel: +90-312-440-0333; Fax: +90-312-438-2792; E-mail: egecanserefoglu @hotmail.com Conflict of Interest: Ege Can Serefoglu and Wayne J.G. Hellstrom are consultants for Allergan Inc., Irwine, CA, USA.
Statement of Authorship
Category 1 (a) Conception and Design Ege C. Serefoglu; Elin M. Grissom; Wayne R. Hawley; Gary P. Dohanich (b) Acquisition of Data Wayne R. Hawley; George F. Lasker; Suresh C. Sikka; Elin M. Grissom; Sree H. Mandava (c) Analysis and Interpretation of Data Ege C. Serefoglu; Wayne R. Hawley J Sex Med **;**:**–**
6
Category 2 (a) Drafting the Article Ege C. Serefoglu; Wayne R. Hawley (b) Revising It for Intellectual Content Gary P. Dohanich; Wayne J.G. Hellstrom
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