Research in pharmacotherapy for erectile dysfunction

Penile erection is a neurovascular phenomenon and multiple pathogenic factors, such as vascular risk factors or diseases, neurologic abnormalities, and hormonal disturbances, are associated with erectile dysfunction (ED) ( 1 ). Although oral phosphodiesterase-5 (PDE5) inhibitors, which enhance the nitric oxide (NO)-cGMP pathway by inhibiting the breakdown of cGMP, are generally regarded as an effective therapy for ED, men with ED from diabetes or radical prostatectomy respond poorly to these drugs ( 2 ). Severe endothelial dysfunction and cavernous nerve damage are mainly responsible for the poor responsiveness of these patients to PDE5 inhibitors ( 3 , 4 ). Because the effects of PDE5 inhibitors rely on endogenous NO formation, PDE5 inhibitors may fail to increase the level of cGMP above the necessary threshold if the endogenous bioavailable NO is insufficient as the result of severe endothelial dysfunction or nerve damage. Moreover, PDE5 inhibitors are not able to reverse the underlying vascular or neural dysfunction associated with ED. It is another limitation that PDE5 inhibitor in combination with any nitrate compound is prohibited because of the risk of hypotensive crisis ( 5 ). Therefore, a new treatment strategy that overcomes the pitfalls of currently available oral PDE5 inhibitors is required. Many researchers have tried to develop novel therapeutics that target alternative molecular pathways. With the introduction of apomorphine hydrochloride (Uproma SL) into ED market, therapeutics with a central mode of action which target dopamine and melanocortin receptors have been developed and their effectiveness on penile erection have been intensively investigated in preclinical models. Meanwhile, a variety of pharmacologic agents with a peripheral mode of action have been also studied. Those specifically target sites upstream of the NO-dependent activation of soluble guanylate cyclase, the Rho-kinase pathway, and the Maxi-K channel. Recently, restoring NO bioavailability by rejuvenating or regenerating cavernous endothelial cells, cavernous nerve, or inhibiting tissue fibrosis secondary to tissue hypoxia from neurovascular dysfunction, was found to be a promising therapeutic strategy for ED, especially in men with severe ED that is unresponsive to PDE5 inhibitors. A variety of preclinical trials targeting therapeutic angiogenesis or neural regeneration as well as anti-fibrosis have been reported. This article addresses the current therapeutic targets for ED under clinical or preclinical development, including pharmacotherapy, protein therapy, and gene therapy.

Therapeutic angiogenesis The penis is a richly vascularized organ, and ED is predominantly a vascular disease ( 17 ). The vascular endothelium plays an important role in the regulation of vascular tone and blood flow ( 18 ). The cavernous endothelium, which is located on the inner surface of the lacunar spaces in the erectile tissue, also has a crucial role in regulating the tone of the underlying smooth muscle and physiologic penile erection ( 19 ). Recently, a link between ED and cardiovascular disease was uncovered, and both diseases were shown to share the same risk factors, including hypercholesterolemia, hypertension, diabetes mellitus, and smoking, with endothelial cell dysfunction being the common denominator between these two conditions ( 20 , 21 ). Therefore, the development of a new therapeutic strategy that reestablishes structural and functional microvasculature in the erectile tissue is needed to treat the underlying conditions of ED and to overcome the limitations of oral PDE5 inhibitors. Many investigators have reported preclinical results of angiogenic growth factor therapy, such as vascular endothelial growth factor (VEGF), angiopoietins, and basic fibroblast growth factor (bFGF) ( Table 2 ). Of those growth factors, VEGF and angiopoietins have been the most extensively studied. Local intracavernous delivery of VEGF gene or protein has been shown to restore erectile function in rat models of ED induced by castration, diabetes, or dyslipidemia ( 22 - 25 ). However, it was reported that VEGF administration can initiate vessel formation in adult animals, but by itself promotes the formation of leaky, immature and unstable vessels ( 34 ), which greatly limits the therapeutic utility of VEGF. In comparison, angiopoietin-1, the ligand of the Tie-2 receptor, is known to generate non-leaky, stable, and mature blood vessels in pre- and postnatal angiogenesis ( 35 ). Angiopoietin-1 also counteracts VEGF-induced inflammation in endothelial cells while having an additive effect on vessel formation ( 36 ). It was recently demonstrated in a rat model of hypercholesterolemic ED that adenovirus-mediated combined gene delivery of angiopoietin-1 and VEGF into the corpus cavernosum produces an additive effect on erectile function through complete restoration of cavernous endothelial cell content compared with that of either therapy alone ( 26 ). Table 2 Preclinical in vivo gene or protein therapies targeting angiogenesis Authors Gene or protein Animal model Duration Efficacy Rogers et al . ( 22 ) AAV-VEGF Castrated rats 1 mo Partial Dall’Era et al . ( 23 ) PEI-VEGF DM rats (STZ) 3 wk Partial Gholami et al . ( 24 ) VEGF protein Dyslipidemic rats 4 mo Partial Yamanaka et al . ( 25 ) VEGF protein DM rats (STZ) 6 wk Complete Ryu et al . ( 26 ) Ad-Ang1 + Ad-VEGF Dyslipidemic rats 8 wk Complete Ryu et al . ( 27 ) Ad-COMP-Ang1 Dyslipidemic mice 8 wk Complete Jin et al . ( 28 ) Ad-COMP-Ang1 DM mice (STZ) 4 wk Complete COMP-Ang1 protein DM mice (STZ) 4 wk Complete Jin et al . ( 29 ) COMP-Ang1 protein db/db mice 2 wk Complete Kwon et al . ( 30 ) Ang4 protein DM mice (STZ) 1 wk Partial Kwon et al . ( 31 ) Apelin protein Dyslipidemic mice 1 d Partial Bae et al . ( 32 ) bFGF hydrogel CNI rats 4 wk Partial Nishimatsu et al . ( 33 ) Ad-adrenomedullin + Ad-Ang1 DM rats (STZ) 4 wk Complete AAV, adeno-associated virus; Ad, adenovirus; Ang1, angiopoietin-1; Ang4, angiopoietin-4; bFGF, basic fibroblast growth factor; CNI, cavernous nerve injury; COMP, cartilage oligomeric matrix protein; DM, diabetes mellitus; PEI, polyethyleneimine; STZ, streptozotocin; VEGF, vascular endothelial growth factor. It was also reported in mouse models of diabetic ED and dyslipidemic ED that a single intracavernous injection of adenovirus-mediated synthetic angiopoietin-1 gene, a soluble and potent angiopoietin-1 variant, or two successive intracavernous injections of synthetic angiopoietin-1 protein significantly increased cavernous endothelial cell proliferation, endothelial nitric oxide synthase (eNOS) phosphorylation, and cGMP expression and decreased the production of reactive oxygen species (ROS), such as superoxide anion and peroxynitrite. These changes restored erectile function up to 4 weeks in diabetic mice and 8 weeks in dyslipidemic mice ( 27 - 29 ). Although relatively long-term recovery of erectile function achieved with angiopoietin-1 protein is noteworthy, difficulty in large-scale production of the recombinant protein limits the clinical application of this therapy. Angiopoietin-4 also represents a family of angiopoietins and plays a crucial role in angiogenesis ( 37 ). It was found that local delivery of angiopoietin-4 protein restores the endogenous Akt-NO pathway through regeneration of cavernous endothelial cells and inhibition of ROS generation, which resulted in recovery of erectile function in diabetic mice. However, the effect of angiopoietin-4 protein on diabetes-induced ED was partial, and the duration of erectile functio

Up-regulation of profibrotic factors and cavernous fibrosis from increased collagen synthesis is also an important cause of ED. Among the various profibrotic factors, Transforming growth factor-β (TGF-β) has been suggested as the most relevant fibrogenic cytokine. TGF-β1 expression is known to be up-regulated in the corpus cavernosum tissues of STZ-induced diabetic rats and in mice with CNI ( 57 , 58 ). Moreover, it was reported that there is an increase in cavernous TGF-β1 expression and collagen synthesis in patients with ED from vascular risk factors or spinal cord injury ( 59 , 60 ). TGF-β1 is known to mediate its fibrotic effects by activating the receptor-associated Smads, including Smad2 and Smad3 ( 61 ). TGF-β1 binds to a type I receptor known as activin receptor-like kinase (ALK) 5. The phosphorylation of serine/threonine residues in ALK5 subsequently phosphorylates the major downstream signaling molecules Smad2 and Smad3. When phosphorylated, Smad2/3 form a heteromeric complex with Smad4, which translocates into the nucleus and regulates the transcription of TGF-β1-responsive genes, thereby inducing fibrosis-related changes ( 61 ). Moreover, TGF-β-Smad2/3 signal pathway is known to mediate smooth muscle cell apoptosis and to inhibit endothelial cell proliferation. It was recently observed that apoptosis in cavernous smooth muscle and endothelial cells and up-regulation of the TGF-β1-Smad2 pathway in the corpus cavernosum of the mouse begin as early as 3 days after CNI ( 58 ). TGF-β1-Smad2/3 signal pathway in the penis was also activated in both diabetic rats and men with spinal cord injury ( 57 , 60 ). These findings suggest that inhibition of TGF-β signaling pathway might be efficacious in the treatment of ED. Preclinical studies targeting anti-fibrosis are summarized in Table 4 . Table 4 Preclinical in vivo gene or protein therapies targeting anti-fibrosis Authors Gene or protein Animal model Duration Efficacy Song et al . ( 62 ) Ad-Smad7 CNI mice 2 wk Partial Li et al . ( 63 ) TGF-β1 antagonist peptide (P144) DM rats (STZ) 4 wk Partial Ad, adenovirus; TGF-β1, transforming growh factor-β1. Smad7 is an inhibitory Smad protein that blocks TGF-β1 signaling through a negative feedback loop. Smad7 protein binds to TGF-β1 type I receptor, which blocks the phosphorylation of the receptor-associated Smads, including Smad2 and Smad3, and eventually inhibits down-stream signaling of TGF-β1 ( 64 ). Thus, Smad7 inhibits diverse cellular processes regulated by TGF-β1, such as cell proliferation, apoptosis, and fibrosis ( 64 ). It was recently reported that a single injection of an adenovirus encoding the Smad7 gene into the corpus cavernosum of CNI mice significantly decreased the production of extracellular matrix proteins, including PAI-1, fibronectin, collagen I, and collagen IV; decreased endothelial cell apoptosis; and induced eNOS phosphorylation, and that these changes were associated with physiologically relevant changes in erectile function ( 62 ). PT144 peptide is a TGF-β antagonist peptide comprised of amino acids derived from the TGF-β type III receptor and is known to have anti-fibrotic activity in animal models of fibrotic diseases. It was reported in STZ-induced diabetic rats that intraperitoneal administration of PT144 decreased cavernous fibrosis and restored erectile function ( 63 ). These findings suggest that an anti-fibrotic strategy through inhibition of the TGF-β signaling pathway may represent a promising therapeutic strategy for ED from CNI or diabetes.