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MP43-18 EARLY REGULAR TREATMENT WITH A CANNABINOID 2 RECEPTOR AGONIST PROTECTED ERECTILE FUNCTION IN A RAT MODEL OF CAVERNOUS NERVE CRUSH INJURY
THE JOURNAL OF UROLOGYâ
e484
MP43-16 CAROTID ATHEROSCLEROSIS EXPRESSED BY INCREASED INTIMA MEDIA THICKNESS IS ASSOCIATED WITH LOW ADHERENCE TO MEDITERRANEAN DIET IN ERECTILE DYSFUNCTION PATIENTS A. Rempelakos*, Ch Vlachopoulos, K. Makarounis, N. Ioakimidis, A. Katevatis, Athens, Greece; A. Aggelis, Athens, Greece; D. Karagiannis, Ch Fasoulakis, Athens, Greece INTRODUCTION AND OBJECTIVES: The Mediterranean type of diet, rich in fruits, legumes, vegetables and nuts, is a healthy dietary pattern, gaining widely recognition as a non-pharmaceutical mean for cardiovascular disease prevention due to its antioxidant components and anti-inflammatory properties. Endothelial dysfunction and subclinical inflammation are important pathophysiologic mechanisms underlying both vasculogenic erectile dysfunction (ED) and atherosclerosis in other vascular beds. Increased carotid IMT (>0,9mm) relates to traditional risk factors and associates with an unfavorable cardiovascular outcome. Purpose of our study is to investigate the association of Mediterranean type of diet with structural changes in large arteries in ED patients, a relation which has not been defined yet. METHODS: Forty-five ED patients (aged 5611 years) underwent carotid ultrasound for evaluation of intima medial thickness (IMT) in the common carotid artery. Overall assessment of dietary habits was evaluated through a special diet score (the Med-DietScore, theoretical range 0e55), which assesses adherence to the Mediterranean dietary pattern. Higher values on the score indicate greater adherence to this pattern and, consequently, healthier dietary habits. RESULTS: Med-diet score was significantly associated with age (r¼-0.215, P<0.05) systolic and pulse pressure (r¼-0.198 and r¼-0.277, respectively, all P<0.05). In univariate analysis Med-DietScorewas inversely associated with carotid IMT (r¼-0.43, P<0.001, figure). In order to further evaluate the impact of Mediterranean diet on carotid wall thickness, multiple linear regression analysis was applied, which revealed that MedDiet Score was inversely associated with IMT after adjustment for history and treatment of hypertension, hypercholesterolemia, diabetes mellitus, as well as use of statinsand smoking (b ¼ -0.305, p<0.01). Patients with a mean IMT >0.9 mmhad significantly lower Med-Diet Score as compared to subjects with lower IMT values (274 vs 335, P<0.05). CONCLUSIONS: The inverse association between Med-Diet Score and carotid IMT indicates an unhealthy dietary life style in ED patients that contributes to an adverse cardiovascular profile and may help in preventing further vascular damage by adapting healthier dietary habits. Source of Funding: NONE
MP43-17 SONIC HEDGEHOG REGULATION OF COLLAGEN IN THE PENIS Christopher Bond, Chicago, IL; Kevin McVary, Springfield, IL; Carol Podlasek*, Chicago, IL INTRODUCTION AND OBJECTIVES: Cavernous nerve (CN) injury, which occurs during prostatectomy, decreases penile smooth muscle through an apoptotic mechanism, leading to erectile dysfunction (ED). As smooth muscle is lost, collagen production increases and there is a change in subtypes. This occurs in ED patients by a largely undefined mechanism. We’ve shown in previous studies that the sonic hedgehog (SHH) pathway is critical for the response of the penis to denervation. It has recently been suggested that SHH may play a role in renal and pulmonary fibrosis and collagen production during lung embryogenesis. Our preliminary studies suggest that collagen production is responsive to SHH signaling and we hypothesize that SHH may be a regulator of collagen in the penis.
Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014
METHODS: Collagen abundance was quantified by trichrome stain and Image J analysis in corpora cavernosal tissue of prostatectomy, diabetic and control (Peyronnie’s) patients, and in rat penis from: 1.) CN crushed, 2.) SHH inhibited, 3.) SHH treated control and CN injured penis, and 4.) Postnatal development time points. RESULTS: Our results show that collagen was increased 16% in prostatectomy and 20% in diabetic patients. SHH treatment of the penis at the time of CN injury prevented collagen induction by 20% at 2 days and 7% at 4 days after injury, as SHH became depleted from the delivery vehicle. During the first week after birth smooth muscle is abundant with minimal collagen synthesis. Collagen abundance increased significantly between postnatal day 7 and 13. CONCLUSIONS: These results show that SHH suppresses collagen induction in response to CN injury. This is an innovative idea with significant potential for intervention and suggests that the SHH pathway sits at the nexus of several key pathways which regulate erectile function. Source of Funding: NIH/NIDDK DK-079184
MP43-18 EARLY REGULAR TREATMENT WITH A CANNABINOID 2 RECEPTOR AGONIST PROTECTED ERECTILE FUNCTION IN A RAT MODEL OF CAVERNOUS NERVE CRUSH INJURY Ettore Di Trapani*, Colicchia Michele, Alberto Briganti, Manuela Tutolo, Maria Passoni, Francesco Montorsi, Fabio Benigni, Niccolo Petter Hedlund, Milan, Italy INTRODUCTION AND OBJECTIVES: Despite the introduction of nerve-sparing radical prostatectomy (NSRP), erectile dysfunction (ED) is a common and challenging sequele. The ED in NSRP is caused by neurapraxia that causes penile hypoxia, loss of nerves, smooth muscle fibrosis and veno-occlusive dysfunction. Recent studies reported immunomodulatory and antifibrotic activity of cannabinoids. We aimed to test the effect of a cannabinoid-2 receptor agonist in a rat model for pelvic neurapraxia METHODS: After ethical approval, Sprague Dawley rats (n¼30, 250-300 g) were A) sham-operated (n¼10); or subjected to bilateral cavernous-nerve (CN) crush injury (CNI) and B) treated with JWH-133 (1mg/kg, intraperitoneal, N¼12), or C) vehicle (intraperitoneal, N¼12). Treatment was initiated 3 days before surgery until 7 days thereafter. Mean arterial blood pressure (MAP) was measured by catheterizing carotid artery and erectile function by direct measurements of intracavernous pressure (ICP) during electrical stimulation (2, 4 and 6V) of the CN. Statistical analysis was performed using an ANOVA (Newman Keuls). Values are meanSEM. RESULTS: Basal ICP was similar in the 3 groups and amounted to 162.2, 223.2, and 181.8cmH2O for sham, CNI + vehicle, and CNI + JWH-133, respectively. MAP amounted to 1338 (sham), 1378 (CNI + vehicle), and 1259cmH2O (CNI+JWH-133). Vehicle treated rats with CNI exhibited at any stimulation parameter significantly lower erectile responses recorded as ICP/MAP than both sham and CNI rats treated with JWH-133. The ICP/MAP values for 2, 4, and 6V were 34.44.2, 42.34.7, and 46.35.4 for vehicle treated CNI rats (p<0.05 vs sham and JWH-133). Corresponding data for sham were 62.55, 704.3, and 75.17.1. ICP/MAP in CNI JWH133 treated rats were not different from sham and amounted to 51.45.4, 74.610, and 87.211.8cmH2O/second. Corresponding AUC values for CNI rats treated with vehicle were lower (p<0.01-0.05) and amounted to 10.51.9, 14.61.9, and 17.92.5cmH2O/second. CNI rats treated with JWH-133 also exhibited lower AUC than sham (p<0.05) with values amounting to 195.4, 31.46, and 344.8cmH2O/second. At 4 and 6V, the AUC values of JWH-133treated CNI rats were higher (p<0.05) than those of vehicle-treated CNI rats.
THE JOURNAL OF UROLOGYâ
Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014
e485
CONCLUSIONS: Early initiated perioperative regular treatment for 10 days with the CB2 agonist JWH-133 restored maximal ICP during erections in rats with CNI but only partially improved the total erectile responses recorded as the AUC. Source of Funding: none
MP43-19 EFFECT OF PHARMACOLOGICAL POST CONDITIONING ON ISCHEMIA-REPERFUSION INJURY IN THE RAT TESTIS Motoaki Saito*, Nankoku, Japan; Shogo Shimizu, Panagiota Tsounapi, Yonago, Japan; Takahiro Shimizu, Nankoku, Japan; Masashi Honda, Takehiro Sejima, Atsushi Takenaka, Shuhei Tomita, Yonago, Japan INTRODUCTION AND OBJECTIVES: The effects of administered KATP channel openers or blockers during ischemia are still controversial in many organs/tissues. To examine whether the administration of KATP channel openers or blockers before or during ischemia ameliorates IR injury in the testis. METHODS: Eight-week-old male SD rats were subjected to 2 h right testicular ischemia followed by 24 h reperfusion. The selective mitochondrial (mito) KATP channel blocker, 5-hydroxydecanoate (5-HD) (40 mg/kg), non-selective KATP channel blocker glibenclamide (Glc) (5 mg/kg) or selective mito KATP channel opener diazoxide (DZ) (10 mg/kg), non-selective KATP channel opener cromakalim (Crom) (300 mg/kg) were administered intraperitoneally 15 min prior to the ischemia or 45 min before the induction of reperfusion. Tissue damage was evaluated by malondialdehyde concentration, myeloperoxidase activity, histological evaluation and TUNEL assay in the testis. RESULTS: There was a significant reduction in oxidative stress, neutrophil infiltration, histological score and apoptosis in the IR model in the testis when DZ and Crom were administrated before ischemia. However, this reduction was not seen with the administration of DZ or Crom during ischemia. In addition, there was a significant reduction in the testicular IR injury with the administration of Glc, but not 5-HD during ischemia. CONCLUSIONS: The pre-conditioning effect on IR injury in the testis was confirmed by treatment with DZ. The administration of non-selective KATP channel blocker Glc during ischemia ameliorated against the testicular IR injury. On the other hand, the selective mito KATP channel blocker, 5-HD and KATP channel openers during ischemia did not reduce the testicular IR injury. These data suggest that opening of KATP channel before ischemia, and blocking of the sarcolemmal KATP channel during the testicular ischemia may have a protective effect. Glc could be an effective drug to manage the post-ischemic injury after testicular torsiondetorsion injury.
Source of Funding: A grant in aid from the Ministry of Education, Science, and Culture of Japan (#23118)
MP43-20 NANOTECHNOLOGY-BASED IMPLANT FOR LONG TERM TESTOSTERONE REPLACEMENT Eugenia Nicolov, Silvia Ferrati, Houston, TX; Randy Goodall, Lee Hudson, Sharath Hosali, Michael Crowley, Austin, TX; Ganesh Palapattu, Ann Arbour, MI; Mohit Khera*, Alessandro Grattoni, Houston, TX INTRODUCTION AND OBJECTIVES: Current delivery approaches in testosterone replacement therapy (TRT) generate fluctuations in testosterone plasma concentration. The objective of this study was to develop a constant, long-term TRT method. We have developed the implantable Personalized Molecular Drugdelivery System (PMDS) for the constant delivery of therapeutic agents using a silicon-based nanofluidics chip with nanochannels (density over 10,000,000/cm2) to control the rate of drug release via spatial and electrostatic nano-confinement with fabricated channel cross-section heights ranging from 2.5 to 250 nm. The sustained release is realized by constrained diffusion where the channel dimensions are precisely determined to create a steady diffusion of molecules. Here we demonstrate the use of the PMDS device to successfully delivery testosterone with long term constant plasma levels in rats. METHODS: In this study, the subcutaneous PMDS implant is made of machined titanium (see Figure) and contains the silicon-based nanofluidic chip, which is fabricated in a leading semiconductor foundry and acts as the rate controlling window. In vivo study: Three different testosterone formulations and a placebo formulation were aseptically loaded into the sterilized capsules and implanted into castrated male Sprague-Dawley rats (7-8 month old). A group of castrated rats received approximately 1/3 of a conventional testosterone pellet. A total of 54 rats were used including an intact control group. Blood samples were collected periodically for 126 days from the saphenous vein. Serum testosterone levels were measured by mass spectrometry. In vitro release of the formulations from identical capsules was measured in parallel by HPLC. RESULTS: The Figure shows the constant release of one testosterone formulation for 126 days. Intact and placebo serum testosterone levels are shown. CONCLUSIONS: The PMDS implant delivers a relatively constant dose and mitigates the serum testosterone variability associated with implantable TRT modalities and will become an attractive alternative strategy for the long-term treatment of male hypogonadism.
e484
MP43-16 CAROTID ATHEROSCLEROSIS EXPRESSED BY INCREASED INTIMA MEDIA THICKNESS IS ASSOCIATED WITH LOW ADHERENCE TO MEDITERRANEAN DIET IN ERECTILE DYSFUNCTION PATIENTS A. Rempelakos*, Ch Vlachopoulos, K. Makarounis, N. Ioakimidis, A. Katevatis, Athens, Greece; A. Aggelis, Athens, Greece; D. Karagiannis, Ch Fasoulakis, Athens, Greece INTRODUCTION AND OBJECTIVES: The Mediterranean type of diet, rich in fruits, legumes, vegetables and nuts, is a healthy dietary pattern, gaining widely recognition as a non-pharmaceutical mean for cardiovascular disease prevention due to its antioxidant components and anti-inflammatory properties. Endothelial dysfunction and subclinical inflammation are important pathophysiologic mechanisms underlying both vasculogenic erectile dysfunction (ED) and atherosclerosis in other vascular beds. Increased carotid IMT (>0,9mm) relates to traditional risk factors and associates with an unfavorable cardiovascular outcome. Purpose of our study is to investigate the association of Mediterranean type of diet with structural changes in large arteries in ED patients, a relation which has not been defined yet. METHODS: Forty-five ED patients (aged 5611 years) underwent carotid ultrasound for evaluation of intima medial thickness (IMT) in the common carotid artery. Overall assessment of dietary habits was evaluated through a special diet score (the Med-DietScore, theoretical range 0e55), which assesses adherence to the Mediterranean dietary pattern. Higher values on the score indicate greater adherence to this pattern and, consequently, healthier dietary habits. RESULTS: Med-diet score was significantly associated with age (r¼-0.215, P<0.05) systolic and pulse pressure (r¼-0.198 and r¼-0.277, respectively, all P<0.05). In univariate analysis Med-DietScorewas inversely associated with carotid IMT (r¼-0.43, P<0.001, figure). In order to further evaluate the impact of Mediterranean diet on carotid wall thickness, multiple linear regression analysis was applied, which revealed that MedDiet Score was inversely associated with IMT after adjustment for history and treatment of hypertension, hypercholesterolemia, diabetes mellitus, as well as use of statinsand smoking (b ¼ -0.305, p<0.01). Patients with a mean IMT >0.9 mmhad significantly lower Med-Diet Score as compared to subjects with lower IMT values (274 vs 335, P<0.05). CONCLUSIONS: The inverse association between Med-Diet Score and carotid IMT indicates an unhealthy dietary life style in ED patients that contributes to an adverse cardiovascular profile and may help in preventing further vascular damage by adapting healthier dietary habits. Source of Funding: NONE
MP43-17 SONIC HEDGEHOG REGULATION OF COLLAGEN IN THE PENIS Christopher Bond, Chicago, IL; Kevin McVary, Springfield, IL; Carol Podlasek*, Chicago, IL INTRODUCTION AND OBJECTIVES: Cavernous nerve (CN) injury, which occurs during prostatectomy, decreases penile smooth muscle through an apoptotic mechanism, leading to erectile dysfunction (ED). As smooth muscle is lost, collagen production increases and there is a change in subtypes. This occurs in ED patients by a largely undefined mechanism. We’ve shown in previous studies that the sonic hedgehog (SHH) pathway is critical for the response of the penis to denervation. It has recently been suggested that SHH may play a role in renal and pulmonary fibrosis and collagen production during lung embryogenesis. Our preliminary studies suggest that collagen production is responsive to SHH signaling and we hypothesize that SHH may be a regulator of collagen in the penis.
Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014
METHODS: Collagen abundance was quantified by trichrome stain and Image J analysis in corpora cavernosal tissue of prostatectomy, diabetic and control (Peyronnie’s) patients, and in rat penis from: 1.) CN crushed, 2.) SHH inhibited, 3.) SHH treated control and CN injured penis, and 4.) Postnatal development time points. RESULTS: Our results show that collagen was increased 16% in prostatectomy and 20% in diabetic patients. SHH treatment of the penis at the time of CN injury prevented collagen induction by 20% at 2 days and 7% at 4 days after injury, as SHH became depleted from the delivery vehicle. During the first week after birth smooth muscle is abundant with minimal collagen synthesis. Collagen abundance increased significantly between postnatal day 7 and 13. CONCLUSIONS: These results show that SHH suppresses collagen induction in response to CN injury. This is an innovative idea with significant potential for intervention and suggests that the SHH pathway sits at the nexus of several key pathways which regulate erectile function. Source of Funding: NIH/NIDDK DK-079184
MP43-18 EARLY REGULAR TREATMENT WITH A CANNABINOID 2 RECEPTOR AGONIST PROTECTED ERECTILE FUNCTION IN A RAT MODEL OF CAVERNOUS NERVE CRUSH INJURY Ettore Di Trapani*, Colicchia Michele, Alberto Briganti, Manuela Tutolo, Maria Passoni, Francesco Montorsi, Fabio Benigni, Niccolo Petter Hedlund, Milan, Italy INTRODUCTION AND OBJECTIVES: Despite the introduction of nerve-sparing radical prostatectomy (NSRP), erectile dysfunction (ED) is a common and challenging sequele. The ED in NSRP is caused by neurapraxia that causes penile hypoxia, loss of nerves, smooth muscle fibrosis and veno-occlusive dysfunction. Recent studies reported immunomodulatory and antifibrotic activity of cannabinoids. We aimed to test the effect of a cannabinoid-2 receptor agonist in a rat model for pelvic neurapraxia METHODS: After ethical approval, Sprague Dawley rats (n¼30, 250-300 g) were A) sham-operated (n¼10); or subjected to bilateral cavernous-nerve (CN) crush injury (CNI) and B) treated with JWH-133 (1mg/kg, intraperitoneal, N¼12), or C) vehicle (intraperitoneal, N¼12). Treatment was initiated 3 days before surgery until 7 days thereafter. Mean arterial blood pressure (MAP) was measured by catheterizing carotid artery and erectile function by direct measurements of intracavernous pressure (ICP) during electrical stimulation (2, 4 and 6V) of the CN. Statistical analysis was performed using an ANOVA (Newman Keuls). Values are meanSEM. RESULTS: Basal ICP was similar in the 3 groups and amounted to 162.2, 223.2, and 181.8cmH2O for sham, CNI + vehicle, and CNI + JWH-133, respectively. MAP amounted to 1338 (sham), 1378 (CNI + vehicle), and 1259cmH2O (CNI+JWH-133). Vehicle treated rats with CNI exhibited at any stimulation parameter significantly lower erectile responses recorded as ICP/MAP than both sham and CNI rats treated with JWH-133. The ICP/MAP values for 2, 4, and 6V were 34.44.2, 42.34.7, and 46.35.4 for vehicle treated CNI rats (p<0.05 vs sham and JWH-133). Corresponding data for sham were 62.55, 704.3, and 75.17.1. ICP/MAP in CNI JWH133 treated rats were not different from sham and amounted to 51.45.4, 74.610, and 87.211.8cmH2O/second. Corresponding AUC values for CNI rats treated with vehicle were lower (p<0.01-0.05) and amounted to 10.51.9, 14.61.9, and 17.92.5cmH2O/second. CNI rats treated with JWH-133 also exhibited lower AUC than sham (p<0.05) with values amounting to 195.4, 31.46, and 344.8cmH2O/second. At 4 and 6V, the AUC values of JWH-133treated CNI rats were higher (p<0.05) than those of vehicle-treated CNI rats.
THE JOURNAL OF UROLOGYâ
Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014
e485
CONCLUSIONS: Early initiated perioperative regular treatment for 10 days with the CB2 agonist JWH-133 restored maximal ICP during erections in rats with CNI but only partially improved the total erectile responses recorded as the AUC. Source of Funding: none
MP43-19 EFFECT OF PHARMACOLOGICAL POST CONDITIONING ON ISCHEMIA-REPERFUSION INJURY IN THE RAT TESTIS Motoaki Saito*, Nankoku, Japan; Shogo Shimizu, Panagiota Tsounapi, Yonago, Japan; Takahiro Shimizu, Nankoku, Japan; Masashi Honda, Takehiro Sejima, Atsushi Takenaka, Shuhei Tomita, Yonago, Japan INTRODUCTION AND OBJECTIVES: The effects of administered KATP channel openers or blockers during ischemia are still controversial in many organs/tissues. To examine whether the administration of KATP channel openers or blockers before or during ischemia ameliorates IR injury in the testis. METHODS: Eight-week-old male SD rats were subjected to 2 h right testicular ischemia followed by 24 h reperfusion. The selective mitochondrial (mito) KATP channel blocker, 5-hydroxydecanoate (5-HD) (40 mg/kg), non-selective KATP channel blocker glibenclamide (Glc) (5 mg/kg) or selective mito KATP channel opener diazoxide (DZ) (10 mg/kg), non-selective KATP channel opener cromakalim (Crom) (300 mg/kg) were administered intraperitoneally 15 min prior to the ischemia or 45 min before the induction of reperfusion. Tissue damage was evaluated by malondialdehyde concentration, myeloperoxidase activity, histological evaluation and TUNEL assay in the testis. RESULTS: There was a significant reduction in oxidative stress, neutrophil infiltration, histological score and apoptosis in the IR model in the testis when DZ and Crom were administrated before ischemia. However, this reduction was not seen with the administration of DZ or Crom during ischemia. In addition, there was a significant reduction in the testicular IR injury with the administration of Glc, but not 5-HD during ischemia. CONCLUSIONS: The pre-conditioning effect on IR injury in the testis was confirmed by treatment with DZ. The administration of non-selective KATP channel blocker Glc during ischemia ameliorated against the testicular IR injury. On the other hand, the selective mito KATP channel blocker, 5-HD and KATP channel openers during ischemia did not reduce the testicular IR injury. These data suggest that opening of KATP channel before ischemia, and blocking of the sarcolemmal KATP channel during the testicular ischemia may have a protective effect. Glc could be an effective drug to manage the post-ischemic injury after testicular torsiondetorsion injury.
Source of Funding: A grant in aid from the Ministry of Education, Science, and Culture of Japan (#23118)
MP43-20 NANOTECHNOLOGY-BASED IMPLANT FOR LONG TERM TESTOSTERONE REPLACEMENT Eugenia Nicolov, Silvia Ferrati, Houston, TX; Randy Goodall, Lee Hudson, Sharath Hosali, Michael Crowley, Austin, TX; Ganesh Palapattu, Ann Arbour, MI; Mohit Khera*, Alessandro Grattoni, Houston, TX INTRODUCTION AND OBJECTIVES: Current delivery approaches in testosterone replacement therapy (TRT) generate fluctuations in testosterone plasma concentration. The objective of this study was to develop a constant, long-term TRT method. We have developed the implantable Personalized Molecular Drugdelivery System (PMDS) for the constant delivery of therapeutic agents using a silicon-based nanofluidics chip with nanochannels (density over 10,000,000/cm2) to control the rate of drug release via spatial and electrostatic nano-confinement with fabricated channel cross-section heights ranging from 2.5 to 250 nm. The sustained release is realized by constrained diffusion where the channel dimensions are precisely determined to create a steady diffusion of molecules. Here we demonstrate the use of the PMDS device to successfully delivery testosterone with long term constant plasma levels in rats. METHODS: In this study, the subcutaneous PMDS implant is made of machined titanium (see Figure) and contains the silicon-based nanofluidic chip, which is fabricated in a leading semiconductor foundry and acts as the rate controlling window. In vivo study: Three different testosterone formulations and a placebo formulation were aseptically loaded into the sterilized capsules and implanted into castrated male Sprague-Dawley rats (7-8 month old). A group of castrated rats received approximately 1/3 of a conventional testosterone pellet. A total of 54 rats were used including an intact control group. Blood samples were collected periodically for 126 days from the saphenous vein. Serum testosterone levels were measured by mass spectrometry. In vitro release of the formulations from identical capsules was measured in parallel by HPLC. RESULTS: The Figure shows the constant release of one testosterone formulation for 126 days. Intact and placebo serum testosterone levels are shown. CONCLUSIONS: The PMDS implant delivers a relatively constant dose and mitigates the serum testosterone variability associated with implantable TRT modalities and will become an attractive alternative strategy for the long-term treatment of male hypogonadism.