ORIGINAL ARTICLE
Effect of avanafil on rat and human corpus cavernosum S. Gur1,2, S. C. Sikka1, E. A. Pankey3, G. F. Lasker3, S. Chandra1, P. J. Kadowitz3 & W. J. G. Hellstrom1 1 Department of Urology, Tulane University Health Sciences Center, New Orleans, LA, USA; 2 Department of Pharmacology, School of Pharmacy, Ankara University, Ankara, Turkey; 3 Department of Pharmacology, Tulane University Health Sciences Center, New Orleans, LA, USA
Keywords Avanafil—cGMP-electrical field stimulation— human and rat corpus cavernosum—nitric oxide—relaxation Correspondence Serap Gur PhD, Department of Urology, Health Sciences Center, Tulane University, 1430 Tulane Avenue, SL-42, New Orleans, LA 70112, USA. Tel.: +(312) 610 3281; Fax: +(504) 988 5059; E-mail: [email protected] or Prof. Dr Serap Gur, Department of Pharmacology, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey. Tel.: +90 (312) 203 3137; Fax: +90 (312) 213 1081; E-mail: [email protected] Accepted: July 29, 2014 doi: 10.1111/and.12344
Summary We compared the activity of a new phosphodiesterase-5 inhibitor (PDE5i) avanafil with sildenafil and tadalafil in human and rat corpus cavernosum (CC) tissues. The effect of avanafil with several inhibitors and electrical field stimulation (EFS) was evaluated on CC after pre-contraction with phenylephrine. With the PDE5i, sildenafil and tadalafil, concentration–response curves were obtained and cyclic guanosine monophosphate (cGMP) levels were measured in tissues. Avanafil induced relaxation with maximum response of 74 5% in human CC. This response was attenuated by NOS inhibitor and soluble guanylate cyclase (sGC) inhibitor. Avanafil potentiated relaxation responses to acetylcholine and EFS in human CC and enhanced SNP-induced relaxation and showed 3-fold increase in cGMP levels. When compared with sildenafil, avanafil and tadalafil were effective at lower concentrations in human CC. In addition, Sprague–Dawley rats underwent in vivo intracavernosal pressure (ICP) and mean arterial pressure (MAP) measurements. Avanafil increased ICP/MAP that was enhanced by SNP and cavernous nerve (CN) stimulation in rat CC tissues. Also avanafil showed maximum relaxation response of 83 7% in rat CC with 3-fold increase in cGMP concentration. Taken together, these results of our in vivo and in vitro studies in human and rat suggest that avanafil promotes the CC relaxation and penile erection via NO-cGMP pathway.
Introduction Millions of men in the United States suffer from erectile dysfunction (ED) (Bacon et al., 2003; Gareri et al., 2014). Among other risk factors, ED has an increased prevalence with age as 40% of men have ED at age 40 and this increases to 65% at aged 65 and older. Phosphodiesterase-5 inhibitors (PDE5i) are an effective therapy for ED, but despite easy oral use, general efficacy and overall tolerability, many men discontinue treatment because of their side effects (Gonzalgo et al., 2003). In early 2012, a novel, potent and selective PDE5i avanafil (Stendraâ; VIVUS, Inc., Mountain View, CA, USA) was approved by the Food and Drug Administration (FDA). This agent exhibits a favourable pharmacokinetic and pharmacodynamic profile with excellent tolerability and few adverse events when used for the treatment of ED (Zhao et al., 2012). In addition, avanafil displays superior selectivity for PDE5 when compared to other PDE isozymes (Wang et al., 2012) © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 897–903
and has a faster onset of action, acting within 15 min of ingestion. This may give avanafil advantages over the other PDE5i (sildenafil, vardenafil and tadalafil). It has also been reported that avanafil allowed for successful vaginal penetration, intercourse and a change in score on the erectile function (EF) domain (Mulhall et al., 2013). Erection is due to relaxation of smooth muscles both corpus cavernosum (CC) and penile arteries resulting in increased blood flow. In this process, nitrergic nerves and endothelial cells release nitric oxide (NO) which stimulates soluble guanylate cyclase (sGC) in cavernosal smooth muscle resulting in increased synthesis of cyclic guanosine monophosphate (cGMP) (Toda & Okamura, 2003; Andersson, 2011). PDE5i prolong or enhance the effect of NO signalling by inhibiting cGMP breakdown. The goal of this study was to compare such effects of avanafil and other PDE5i at cellular levels using in human CC strips in vitro and also in vivo and in vitro studies in rat. 897
Avanafil-induced relaxation of corpus cavernosum
Materials and methods Human corpus cavernosum A small piece of cavernosal tissue (n = 20) was obtained with consent under institutional review board guidelines from patients (age 43–69 years, mean: 60.4 2.9) with ED and/or Peyronie’s disease who underwent penile prosthesis surgery. Human corpus cavernosum (HCC) tissue sample was placed in cold Krebs solution and immediately transported to the laboratory for in vitro experiments. Fifteen samples were obtained from patients with organic impotence, two samples from patients after radical prostatectomy and three samples from patients with Peyronie’s disease.
S. Gur et al.
SD9 Stimulator. A rest period of at least 10 min was allowed between consecutive nerve stimulations. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC). Rat CC strip preparation Sprague–Dawley adult male rats (300–350 g, n = 24; Harlan, Indianapolis, IN, USA) were anaesthetised with sodium thiobutabarbital (salt hydrate Inactinâ, C-III 100 mg kg 1, i.p.), exsanguinated via the carotid artery and the penis removed. Cavernosal strips (1 9 1 9 8 mm) from the penis were prepared following dissection of the tunica albuginea and surrounding connective tissue and placed in Krebs isotonic solution for further in vitro experiments.
In vivo rat experiments Adult male Sprague–Dawley rats (313–348 g, n = 6) were used in experiments to measure erectile responses. The rats were anaesthetised (Inactin, 100 mg kg 1, i.p.), the trachea was cannulated with polyethylene (PE)-240 tubing to maintain the airway, and the carotid artery was cannulated with PE-50 tubing connected to a transducer (Namic Preceptor, Boston Scientific, Natic, MA, USA) attached to a data acquisition system (Biopac MP 100 System, Santa Barbara, CA, USA) for measurement of mean arterial pressure (MAP). A 25-gauge needle filled with 250 U ml 1 of heparin and connected to PE-50 tubing was inserted into the right crura of the penis and connected to a pressure transducer to measure intracavernosal pressure (ICP). The left jugular vein was catheterised with PE-50 tubing for the systemic administration of drugs and fluids. A heparin filled 25-gauge needle was placed in the right crura of the penis for intracavernosal (IC) administration of drugs. Maximal ICP in response to IC injection of the vasodilator agents or in response to cavernosal nerve (CN) stimulation was measured at the peak of the pressure increase. The area under the curve (AUC) and duration of the increase in ICP were measured to characterise the erectile response. All injections were given in a total volume of 150 ll, and a washout period of 15–30 min was observed between injections. CN stimulation in a separate group of rats was carried out as previously described in the literature (Gur et al., 2008, 2010b; Lasker et al., 2013). For nerve stimulation, the bladder and prostate were exposed through a midline abdominal incision, the CN was identified posterolaterally to the prostate on one side, and a stainless steel bipolar stimulating electrode was placed on the nerve. The CN was stimulated with square-wave pulses at a frequency of 16 Hz with a pulse width of 5 ms and a duration of 60 s at voltages of 2.5, 5.0 and 7.5 V with a Grass Instruments 898
Isometric tension measurements using human and rat in CC strips Three to four cavernosal tissue strips (1 9 1 9 8 mm) were prepared and placed in Krebs isotonic solution consisting of NaCl, 118 mM; NaHCO3, 25 mM; glucose, 5.6 mM; KCl, 4.7 mM; KH2PO4, 1.2 mM; MgSO4 7H2O, 1.17 mM and CaCl2 2H2O, 2.5 mM. The cavernosal tissue strips were mounted under 1 g tension in 20-ml organ bath chambers (Radnoti; Radnoti Glass Technology Inc, Monrovia, CA, USA) containing Krebs solution at 37 °C (pH 7.4) and continuously bubbled with a mixture of 95% O2 and 5% CO2. Tissues strips, with one end attached to an electrode holder and the other to a wire connected to a force transducer, were allowed to equilibrate for 60 min, and the bath medium was changed every 15 min. The CC strips were pre-contracted with phenylephrine (Phe, 10 lM) and allowed to relax after administration of avanafil (10 8–10 3 M). The relaxant response to avanafil was recorded in strips before and after administration of the nonspecific NOS inhibitor (NG-nitro-L-arginine methyl ester, L-NAME, 100 mM, n = 8) and sGC inhibitor (1H-[1,2,4] oxadiazolo [4,3-a]quinoxaline-1-one, ODQ 10 lM, n = 7). The incubation duration with inhibitors was 20 min prior to relaxation curves. In addition, the response to a single concentration of ACh (100 lM) was obtained in Phe-pre-contracted preparations from both rat (n = 10) and human (n = 8) CC tissue in presence of avanafil. Also, effects of avanafil (0.1, 1, 10 and 100 lM) on sodium nitroprusside (SNP)induced relaxation were evaluated. Concentration– response curves for sildenafil (10 8–10 3 M) and tadalafil (10 8–10 3 M) were also obtained for rat (n = 6) and HCC (n = 6) tissue strips. In the nerve stimulation studies, electrical field stimulation (EFS) was first delivered as a train of square-wave © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 897–903
S. Gur et al.
pulses (pulse width, 0.5 ms; intensity, 20 V) at a frequency of 20 Hz across paired platinum electrodes, which were placed on either side of the tissue strips as described previously (Gur et al., 2010a). After Phe-induced precontraction, EFS produced relaxation responses mediated by nitrergic nerves. When a stable level of contraction with Phe was attained, a series of EFS-induced relaxations was elicited in the absence and presence of avanafil (1 lM) in CC tissues from rat (n = 8) and human (n = 8). For EFS response curves, the CC smooth muscle was pre-incubated for 30 min with guanethidine (5 lM) to eliminate noradrenergic influence and atropine (1 lM) to prevent cholinergic responses, respectively. Effect of avanafil on cGMP levels Some of the cavernosal isolated strips from organ bath experiments were rapidly frozen by submerging in liquid nitrogen and stored at 80 °C until cGMP measurements were taken. An ELISA kit (Cat. 581021; Cayman Chemical, Ann Arbor, MI, USA) with 96 wells was used to quantify the cGMP levels. The frozen tissues were crushed into very fine powder and dissolved in 0.1 M HCl and centrifuged as per kit instructions. An aliquot of the supernatant was placed in the wells for cGMP measurements, while another aliquot was used for measurement of protein levels. Both the standard and test wells were incubated with the yellow antibody following kit instructions, and optical density read at 405 nm using the microplate reader. cGMP levels were calculated using the standard curve and expressed as pmol mg 1 protein.
Avanafil-induced relaxation of corpus cavernosum
using computer assisted nonlinear curve fitting analysis from concentration–response curves (Table 1). A P value
Effect of avanafil on rat and human corpus cavernosum S. Gur1,2, S. C. Sikka1, E. A. Pankey3, G. F. Lasker3, S. Chandra1, P. J. Kadowitz3 & W. J. G. Hellstrom1 1 Department of Urology, Tulane University Health Sciences Center, New Orleans, LA, USA; 2 Department of Pharmacology, School of Pharmacy, Ankara University, Ankara, Turkey; 3 Department of Pharmacology, Tulane University Health Sciences Center, New Orleans, LA, USA
Keywords Avanafil—cGMP-electrical field stimulation— human and rat corpus cavernosum—nitric oxide—relaxation Correspondence Serap Gur PhD, Department of Urology, Health Sciences Center, Tulane University, 1430 Tulane Avenue, SL-42, New Orleans, LA 70112, USA. Tel.: +(312) 610 3281; Fax: +(504) 988 5059; E-mail: [email protected] or Prof. Dr Serap Gur, Department of Pharmacology, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey. Tel.: +90 (312) 203 3137; Fax: +90 (312) 213 1081; E-mail: [email protected] Accepted: July 29, 2014 doi: 10.1111/and.12344
Summary We compared the activity of a new phosphodiesterase-5 inhibitor (PDE5i) avanafil with sildenafil and tadalafil in human and rat corpus cavernosum (CC) tissues. The effect of avanafil with several inhibitors and electrical field stimulation (EFS) was evaluated on CC after pre-contraction with phenylephrine. With the PDE5i, sildenafil and tadalafil, concentration–response curves were obtained and cyclic guanosine monophosphate (cGMP) levels were measured in tissues. Avanafil induced relaxation with maximum response of 74 5% in human CC. This response was attenuated by NOS inhibitor and soluble guanylate cyclase (sGC) inhibitor. Avanafil potentiated relaxation responses to acetylcholine and EFS in human CC and enhanced SNP-induced relaxation and showed 3-fold increase in cGMP levels. When compared with sildenafil, avanafil and tadalafil were effective at lower concentrations in human CC. In addition, Sprague–Dawley rats underwent in vivo intracavernosal pressure (ICP) and mean arterial pressure (MAP) measurements. Avanafil increased ICP/MAP that was enhanced by SNP and cavernous nerve (CN) stimulation in rat CC tissues. Also avanafil showed maximum relaxation response of 83 7% in rat CC with 3-fold increase in cGMP concentration. Taken together, these results of our in vivo and in vitro studies in human and rat suggest that avanafil promotes the CC relaxation and penile erection via NO-cGMP pathway.
Introduction Millions of men in the United States suffer from erectile dysfunction (ED) (Bacon et al., 2003; Gareri et al., 2014). Among other risk factors, ED has an increased prevalence with age as 40% of men have ED at age 40 and this increases to 65% at aged 65 and older. Phosphodiesterase-5 inhibitors (PDE5i) are an effective therapy for ED, but despite easy oral use, general efficacy and overall tolerability, many men discontinue treatment because of their side effects (Gonzalgo et al., 2003). In early 2012, a novel, potent and selective PDE5i avanafil (Stendraâ; VIVUS, Inc., Mountain View, CA, USA) was approved by the Food and Drug Administration (FDA). This agent exhibits a favourable pharmacokinetic and pharmacodynamic profile with excellent tolerability and few adverse events when used for the treatment of ED (Zhao et al., 2012). In addition, avanafil displays superior selectivity for PDE5 when compared to other PDE isozymes (Wang et al., 2012) © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 897–903
and has a faster onset of action, acting within 15 min of ingestion. This may give avanafil advantages over the other PDE5i (sildenafil, vardenafil and tadalafil). It has also been reported that avanafil allowed for successful vaginal penetration, intercourse and a change in score on the erectile function (EF) domain (Mulhall et al., 2013). Erection is due to relaxation of smooth muscles both corpus cavernosum (CC) and penile arteries resulting in increased blood flow. In this process, nitrergic nerves and endothelial cells release nitric oxide (NO) which stimulates soluble guanylate cyclase (sGC) in cavernosal smooth muscle resulting in increased synthesis of cyclic guanosine monophosphate (cGMP) (Toda & Okamura, 2003; Andersson, 2011). PDE5i prolong or enhance the effect of NO signalling by inhibiting cGMP breakdown. The goal of this study was to compare such effects of avanafil and other PDE5i at cellular levels using in human CC strips in vitro and also in vivo and in vitro studies in rat. 897
Avanafil-induced relaxation of corpus cavernosum
Materials and methods Human corpus cavernosum A small piece of cavernosal tissue (n = 20) was obtained with consent under institutional review board guidelines from patients (age 43–69 years, mean: 60.4 2.9) with ED and/or Peyronie’s disease who underwent penile prosthesis surgery. Human corpus cavernosum (HCC) tissue sample was placed in cold Krebs solution and immediately transported to the laboratory for in vitro experiments. Fifteen samples were obtained from patients with organic impotence, two samples from patients after radical prostatectomy and three samples from patients with Peyronie’s disease.
S. Gur et al.
SD9 Stimulator. A rest period of at least 10 min was allowed between consecutive nerve stimulations. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC). Rat CC strip preparation Sprague–Dawley adult male rats (300–350 g, n = 24; Harlan, Indianapolis, IN, USA) were anaesthetised with sodium thiobutabarbital (salt hydrate Inactinâ, C-III 100 mg kg 1, i.p.), exsanguinated via the carotid artery and the penis removed. Cavernosal strips (1 9 1 9 8 mm) from the penis were prepared following dissection of the tunica albuginea and surrounding connective tissue and placed in Krebs isotonic solution for further in vitro experiments.
In vivo rat experiments Adult male Sprague–Dawley rats (313–348 g, n = 6) were used in experiments to measure erectile responses. The rats were anaesthetised (Inactin, 100 mg kg 1, i.p.), the trachea was cannulated with polyethylene (PE)-240 tubing to maintain the airway, and the carotid artery was cannulated with PE-50 tubing connected to a transducer (Namic Preceptor, Boston Scientific, Natic, MA, USA) attached to a data acquisition system (Biopac MP 100 System, Santa Barbara, CA, USA) for measurement of mean arterial pressure (MAP). A 25-gauge needle filled with 250 U ml 1 of heparin and connected to PE-50 tubing was inserted into the right crura of the penis and connected to a pressure transducer to measure intracavernosal pressure (ICP). The left jugular vein was catheterised with PE-50 tubing for the systemic administration of drugs and fluids. A heparin filled 25-gauge needle was placed in the right crura of the penis for intracavernosal (IC) administration of drugs. Maximal ICP in response to IC injection of the vasodilator agents or in response to cavernosal nerve (CN) stimulation was measured at the peak of the pressure increase. The area under the curve (AUC) and duration of the increase in ICP were measured to characterise the erectile response. All injections were given in a total volume of 150 ll, and a washout period of 15–30 min was observed between injections. CN stimulation in a separate group of rats was carried out as previously described in the literature (Gur et al., 2008, 2010b; Lasker et al., 2013). For nerve stimulation, the bladder and prostate were exposed through a midline abdominal incision, the CN was identified posterolaterally to the prostate on one side, and a stainless steel bipolar stimulating electrode was placed on the nerve. The CN was stimulated with square-wave pulses at a frequency of 16 Hz with a pulse width of 5 ms and a duration of 60 s at voltages of 2.5, 5.0 and 7.5 V with a Grass Instruments 898
Isometric tension measurements using human and rat in CC strips Three to four cavernosal tissue strips (1 9 1 9 8 mm) were prepared and placed in Krebs isotonic solution consisting of NaCl, 118 mM; NaHCO3, 25 mM; glucose, 5.6 mM; KCl, 4.7 mM; KH2PO4, 1.2 mM; MgSO4 7H2O, 1.17 mM and CaCl2 2H2O, 2.5 mM. The cavernosal tissue strips were mounted under 1 g tension in 20-ml organ bath chambers (Radnoti; Radnoti Glass Technology Inc, Monrovia, CA, USA) containing Krebs solution at 37 °C (pH 7.4) and continuously bubbled with a mixture of 95% O2 and 5% CO2. Tissues strips, with one end attached to an electrode holder and the other to a wire connected to a force transducer, were allowed to equilibrate for 60 min, and the bath medium was changed every 15 min. The CC strips were pre-contracted with phenylephrine (Phe, 10 lM) and allowed to relax after administration of avanafil (10 8–10 3 M). The relaxant response to avanafil was recorded in strips before and after administration of the nonspecific NOS inhibitor (NG-nitro-L-arginine methyl ester, L-NAME, 100 mM, n = 8) and sGC inhibitor (1H-[1,2,4] oxadiazolo [4,3-a]quinoxaline-1-one, ODQ 10 lM, n = 7). The incubation duration with inhibitors was 20 min prior to relaxation curves. In addition, the response to a single concentration of ACh (100 lM) was obtained in Phe-pre-contracted preparations from both rat (n = 10) and human (n = 8) CC tissue in presence of avanafil. Also, effects of avanafil (0.1, 1, 10 and 100 lM) on sodium nitroprusside (SNP)induced relaxation were evaluated. Concentration– response curves for sildenafil (10 8–10 3 M) and tadalafil (10 8–10 3 M) were also obtained for rat (n = 6) and HCC (n = 6) tissue strips. In the nerve stimulation studies, electrical field stimulation (EFS) was first delivered as a train of square-wave © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 897–903
S. Gur et al.
pulses (pulse width, 0.5 ms; intensity, 20 V) at a frequency of 20 Hz across paired platinum electrodes, which were placed on either side of the tissue strips as described previously (Gur et al., 2010a). After Phe-induced precontraction, EFS produced relaxation responses mediated by nitrergic nerves. When a stable level of contraction with Phe was attained, a series of EFS-induced relaxations was elicited in the absence and presence of avanafil (1 lM) in CC tissues from rat (n = 8) and human (n = 8). For EFS response curves, the CC smooth muscle was pre-incubated for 30 min with guanethidine (5 lM) to eliminate noradrenergic influence and atropine (1 lM) to prevent cholinergic responses, respectively. Effect of avanafil on cGMP levels Some of the cavernosal isolated strips from organ bath experiments were rapidly frozen by submerging in liquid nitrogen and stored at 80 °C until cGMP measurements were taken. An ELISA kit (Cat. 581021; Cayman Chemical, Ann Arbor, MI, USA) with 96 wells was used to quantify the cGMP levels. The frozen tissues were crushed into very fine powder and dissolved in 0.1 M HCl and centrifuged as per kit instructions. An aliquot of the supernatant was placed in the wells for cGMP measurements, while another aliquot was used for measurement of protein levels. Both the standard and test wells were incubated with the yellow antibody following kit instructions, and optical density read at 405 nm using the microplate reader. cGMP levels were calculated using the standard curve and expressed as pmol mg 1 protein.
Avanafil-induced relaxation of corpus cavernosum
using computer assisted nonlinear curve fitting analysis from concentration–response curves (Table 1). A P value
