Pharmacology of erectile dysfunction in man

Pharmacology & Therapeutics 111 (2006) 400 – 423 www.elsevier.com/locate/pharmthera

Pharmacology of erectile dysfunction in man Giuseppe Cirino a,b,*, Ferdinando Fusco b, Ciro Imbimbo b, Vincenzo Mirone b b

a Dipartimento di Farmacologia Sperimentale, Via Domenico Montesano 49, 8031 Napoli, Italy Centro Interdipartimentale di Ricerca Preclinica e Clinica di Medicina Sessuale (CIRMS) Via Pansini, Napoli, Italy

Abstract Erectile dysfunction (ED) is defined as the consistent or recurrent inability of a man to attain and/or maintain a penile erection sufficient for sexual activity (2nd International Consultation on Sexual Dysfunction-Paris, June 28th – July 1st, 2003). Following the discovery and introduction of sildenafil, research on the mechanisms underlying penile erection has had an enormous boost and many preclinical and clinical papers have been published in the last 5 years. This review is structured in order to give the reader an overview of the clinical and preclinical data available on the peripheral regulation of and the mediators involved in human penile erection. The most widely accepted risk factors for ED are discussed. The article is focused on human data, and the safety and effectiveness of the 3 commercially available Phosphodiesterase-5 (PDE5) inhibitors used to treat ED are also discussed. D 2005 Elsevier Inc. All rights reserved. Keywords: Erectile dysfunction; Risk factors; Peripheral mechanisms; Human preclinical and clinical; PDE5; Peripheral regulation

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiology of penile erection and erectile dysfunction . . . . . . . . . . . . . . . . . . Erectile dysfunction and risk factors . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Cardiovascular diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Smoking. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The mediators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Contraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. The adrenergic system . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Endothelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3. Angiotensin and angiotensin-converting enzyme. . . . . . . . . . . . . 4.1.4. The Rho-kinase pathway. . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Relaxation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. l-Arginine – nitric oxide pathway and guanylyl cyclase/cGMP pathway. 4.2.2. Prostanoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3. Vasoactive intestinal polypeptide. . . . . . . . . . . . . . . . . . . . . Drugs and therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Phosphodiesterase-5 as therapeutic target . . . . . . . . . . . . . . . . . . . . . 5.1.1. Sildenafil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.2. Tadalafil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.3. Vardenafil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author. Dipartimento di Farmacologia Sperimentale via Domenico Montesano 49, 80131 Napoli, Italy. Tel.: +39 81 678442; fax: +39 81 678403. E-mail address: [email protected] (G. Cirino). 0163-7258/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2005.10.011

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5.2.

Intracavernous therapy . . 5.2.1. Prostaglandin E1 5.2.2. Other therapies . 6. Conclusions and future direction. References . . . . . . . . . . . . . .

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1. Introduction Although nitric oxide (NO) was shown to be a key mediator of penile erection in 1992, it was the discovery and introduction of sildenafil that have resulted in the considerable recent attention focused on research on the mechanisms underlying penile erection. A search of PubMed, one of the most popular search engines for biomedical research, using the search term erectile dysfunction (ED) and limiting results to those published in 1997, the year when sildenafil was launched, reveals a dramatic 15-fold steep increase in frequency of papers compared with previous years (Fig. 1), and this includes papers in ‘‘high impact factor’’ peer-reviewed journals such as Nature Medicine and PNAS. In 1997, the International Journal of Impotence Research, the first scientific journal specifically dedicated to research focussing on male sexual function, was launched. Since then this journal has progressively and quickly increased its impact factor. Concomitant with the evident genuine scientific interest, a massive campaign has been launched in the last few years to highlight the importance for men to look after their sexual well-being. Thus, following launch of sildenafil on the market, papers on ED have been published in medical specialist journals, socioeconomic journals, and in general scientific magazines that can be purchased in a newsagent. Such scientific and nonscientific interest in male sexuality has had undoubtedly positive effects. More economic resources have finally been made available for research in this field, and both the common social concept of ED and the mass media language adopted to deal with such a difficult topic have been polished of the special dust of pruriginous curiosity and the almost preposterous mystery that previously dominated the scene. 350

number of papers

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impotence erectile dysfunction

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19 80 -1 99 7 19 98 -2 00 4

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Fig. 1. The number of scientific articles published in peer-reviewed journals from 1980 to 2004. Data have been obtained by using Ferectile dysfunction_ and Fimpotence_ as keywords.

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Finally, thanks to the abovementioned scientific enthusiasm, preclinical and clinical research have started to generate new information on erectile function, thus casting new light on penile physiology and pharmacology, resulting in revision of old concepts and proposal of new biochemical mediators and pathways. Several clinical reports have revealed a tight correlation between ED and cardiovascular disease (CVD), to the extent that 1 paper invited readers to consider ‘‘The penis as a barometer of endothelial health’’ (McCullough, 2003). The main goal of this review is to examine the current understanding of ED and research into its mechanisms through an analysis of the preclinical and clinical bibliography to date with a particular emphasis on the peripheral regulation of the microcirculation and the mediators involved in the human tissue. For preclinical data obtained in experimental animal models, the reader is referred to other reviews (Andersson, 2001; Toda et al., 2005). 2. Physiology of penile erection and erectile dysfunction The ability to get and keep an erection is important to men for a variety of reasons and it is that variety which makes the assessment of erectile dysfunction (ED) so challenging. ED is defined as the consistent or recurrent inability of a man to attain and/or maintain a penile erection sufficient for sexual activity (2nd International Consultation on Sexual Dysfunction-Paris, June 28th – July 1st, 2003). However, while erection for satisfactory sexual experience is important, erection has also deeper symbolic value, related to selfimage, self-esteem, and perceptions of gender role. Erection is a neurovascular reflex event, subject to modification by the central nervous system and endocrine factors. Satisfactory sexual experience is perceived by the mind, not the penis. It is subjective and modified by conscious and unconscious processes. A man’s perceptions of his partner needs and expectations will also have an influence. Thus, erection is the final outcome of a complex integration of signals. It is essentially a spinal reflex that can be initiated by recruitment of penile afferents, but also by visual, olfactory, and imaginary stimuli. Initially a dilatation of the arterioles and arteries cause an increase of blood flow, followed by an expansion of sinusoidal spaces and volume augmentation. Blood inflow causes a volumetric increase of cavernosal bodies as long as the elastic properties of the penis allow it. When a full turgid state is achieved, the tunica albuginea reaches its maximum expansion and limits the increment of diameter and length. Any further inflow of blood, in accordance with the Laplace rule, causes the penis to shift from a turgid to full rigid state.

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Once gathered in the sinusoidal space, the blood is trapped in the corpora cavernosal due to the so-called veno-occlusive mechanism. In fact, the increased arterial inflow and the subsequent increase in volume lead to compression of the subtunical plexuses between the peripheral sinusoids and the tunica albuginea, thus reducing the venous outflow. As result, during the rigid state the inflow and outflow are almost absent and intracavernous pressure is increased to about 100 mmHg. The contraction of ischiocavernous muscles is an adjuvant mechanism that further increases the intracavernosal pressure and penile rigidity, whereas contraction of bulbocavernous muscle reinforces glans erection. In simpler words, penile erection is an example of microcirculation where the vasodilatation coupled to blood engorgement of the corpora cavernosa causes a compression of the vein between the corpora and the albuginea leading to the erection. What makes the physiology of the corpus cavernosum intriguing is the fact that in the flaccid state of the penis there is a predominance of adrenergic tone, as opposed to the erect state during which there is a reduction of adrenergic tone with increased vasodilatation. Thus, the penis needs the erection to be exposed to fresh blood and well oxygenated. Indeed, during the REM phase of sleep, men physiologically experience a certain number of erections that are a signal of normal erectile

function. It is clear then that the balance between contraction and relaxation is necessary also in absence of erotic stimuli or sexual activity, being critical for a correct function of the male organ, providing oxygenation that is indispensable for the maintenance of the healthy status of the corpora cavernosa. This is clearly visible by using the nocturnal penile tumescence (NPT) test (Fig. 2) For example, analysis of the Rigiscan trace allows diagnosis of organic versus psychogenic ED (Fig. 3). The penile erection reflex involves both autonomic and somatic efferents and is modulated by supraspinal influences. Several central transmitters involved in erectile control have been identified. On the one hand, dopamine, acetylcholine, nitric oxide (NO), and peptides such as oxytocin and adrenocorticotropic/a-melanocyte-stimulating hormone seem to have a facilitatory role, whereas encephalins are inhibitory and serotonin may be either facilitatory or inhibitory. Neurogenic NO is considered the most important factor for relaxation of penile vessels and corpus cavernosum. The role of other mediators released from nerves or endothelium has not been definitely established. Dopamine receptors in central nervous centers participating in the initiation of erection have been targeted for the treatment of ED. Apomorphine, administered sublingually, was the first drug of this type to be registered. However, apomorphine was prescribed only briefly due to the

Fig. 2. The Rigiscan: a device for the evaluation of nocturnal penile tumescence. The apparatus records through the ring’s rigidity at the base and tip of the penis.

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strated sensitivity and specificity for detecting treatmentrelated changes in patients with ED (Rosen et al., 1997). Another widely diffused questionnaire is the Sexual Encounter Profile (SEP) that is shown in Fig. 5. IIEF and SEP are currently the most used measures of efficacy in any study evaluating ED treatments. Here follows an analysis of risk factors and the clinical evidence that relate them to ED. 3.1. Age Age is the major risk factor because with aging there are clear changes in the physiology of male sexual activity Table 1 Drugs that can cause erectile dysfunction Antidepressants

Fig. 3. (a) A Rigiscan trace from a nocturnal penile tumescence test from a normal healthy volunteer. The trace shown refers to 1 night. Four erective episodes are visible, with good duration and excellent rigidity both at base and tip of the penis. (b) A Rigiscan trace from a nocturnal penile tumescence test from a severe ED patient. The trace shown is a 1 night record. The absence of erective episodes is suggestive of organic erectile dysfunction.

fact that it caused nausea and vomiting in patients as major side effects.

Antipsychotics

Anthypertensives

3. Erectile dysfunction and risk factors In recent years, several clinical and epidemiologic studies have been performed in order to define the risk factors for ED. ED can be considered epidemic because it is prevalent in excess of normal expectations. The following section will give a survey of the clinical studies that have addressed the possible role played by age, obesity, smoking, and cardiovascular diseases in the development of ED. It is interesting to note that the ED risk factors age, obesity, and smoking are the same risk factors for cardiovascular disease and that there is a literature in both urology and cardiovascular journals on this specific matter. Drugs prescribed for treating specific non-ED diseases for which ED has been reported as side effect represent another risk factor. A summary, by no means exhaustive, is reported in Table 1. In order for the reader to better understand the following part of the review, it is important to know that the primary endpoint in clinical studies on ED is the variation of the International Index of Erectile Function (IIEF) score. The IIEF addresses the relevant domains of male sexual function, that is, erectile function, orgasmic function, sexual desire, intercourse satisfaction, and overall satisfaction, and it has been linguistically validated in 10 languages (Fig. 4). This questionnaire is readily self-administered and has demon-

Drugs of abuse

Anticonvulsants

Miscellaneous

Imipramine Doxepin Trazodone Isocarboxazid Desipramine, protryptiline Maprotiline Nortryptiline Clomipramine Tranylcypromine Amitriptiline Amoxapine Phenelzine Chlorpromazine Thioridazine Pimozide Sulpiride Thiotixene Haloperidol Fluphenazine Hydrochlorothiazide Reserpine Propanolol Guanadrel Spironolattone Guanethidine Chlorthalidone Labetalol Alpha-methyldopa Clonidine Guanabenz Atenolol Alcohol Cocaina MDMA Amphetamines Barbiturates Phenytoin Primidone Carbamazepine Cimetidine Ketamine Metadone Digoxin Lithium Thiabendazole Clofibrate Acetazolamide Disulfiram Ketoconazole

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G. Cirino et al. / Pharmacology & Therapeutics 111 (2006) 400 – 423 International Index of Erectile Function (IIEF): 1. Over the past 4 weeks, how often were you able to get an erection during sexual activity? 2. Over the past 4 weeks, when you had erections with sexual stimulation, how often were your erections hard enough for penetration? 3. Over the past 4 weeks, when you attempted sexual intercourse, how often were you able to penetrate (enter) your partner? 4. Over the past 4 weeks, during sexual intercourse, how often were you able to maintain your erection after you had penetrated (entered) your partner? 5. Over the past 4 weeks, during sexual intercourse, how difficult was it to maintain your erection to completion of intercourse? 6. Over the past 4 weeks, how many times have you attempted sexual intercourse? 7. Over the past 4 weeks, when you attempted sexual intercourse how often was it satisfactory for you? 8. Over the past 4 weeks, how much have you enjoyed sexual intercourse? 9. Over the past 4 weeks, when you had sexual stimulation or intercourse how often did you ejaculate? 10. Over the past 4 weeks, when you had sexual stimulation or intercourse how often did you have the feeling of orgasm or climax (with or without ejaculation)? 11. Over the past 4 weeks, how often have you felt sexual desire? 12. Over the past 4 weeks, how would you rate your level of sexual desire? 13. Over the past 4 weeks, how satisfied have you been with your overall sex life? 14. Over the past 4 weeks, how satisfied have you been with your sexual relationship with your partner? 15. Over the past 4 weeks, how do you rate your confidence that you can get and keep an erection? IIEF score system: Questions 1,2,3,4,7,9: 0= No sexual activity; 1= Almost never or never; 2= A few times (much less than half the time); 3= Sometimes (about half the time); 4= Most times (much more than half the time); 5= Almost always or always. Question 5: 0=Did not attempt intercourse; 1= Not difficult; 2=Slightly difficult; 3= Difficult; 4= Very difficult; 5= Extremely difficult Question 6: 0=No attempts; 1= One to two attempts, 2= Three to four attempts; 3= Five to six attempts; 4= Seven to ten attempts; 5= Eleven or more attempts Question 8: 0=No intercourse; 1=Not enjoyable; 2= Not very enjoyable; 3= Fairly enjoyable; 4= Highly enjoyable; 5= Very highly enjoyable Question 11: 1= Almost never or never; 2= A few times; 3= Sometimes; 4= Most times; 5= almost always or always Question 12,15: 1= Very dissatisfied; 2= Moderately dissatisfied; 3= About equally satisfied and dissatisfied; 4= Moderately satisfied; 5= Very satisfied

Fig. 4. The International Index of Erectile Function (IIEF) Questionnaire.

(Lunenfeld, 2002; Seftel, 2003). Indeed, aging causes a lengthening of the excitement phase accompanied by a delayed erection, a lengthening of the plateau phase with a long interval to ejaculation and a decreased penile rigidity. During postorgasm, there is a more rapid detumescence and a longer refractory period. Indeed, in the Massachusetts Male Aging Study (MMAS) on 1290 men within an age range between 40 and 70 years old, there was a reported prevalence of ED of ¨ 53%, strongly suggesting that the variable most associated with ED is age (Feldman et al., 1994). A difficulty in assessing the pathophysiology of the decline of human erectile function with aging lies in the difficult discrimination between aging and concomitant diseases. A Patient Sexual Encounter Profile (SEP) Date of Sexual Encounter ______/______/____ (day / month / year) Time of Sexual Encounter______ 1. Were you able to achieve at least some erection (some enlargement of the penis)? If yes, place 1 in the box and CONTINUE. If no, place 0 in the box and STOP. 2. Were you able to insert your penis into your partner’s vagina? If yes, place 1 in the box. If no, place 0 in the box. 3. Did your erection last long enough for you to have successful intercourse? If yes, place 1 in the box. If no, place 0 in the box. 4. Were you satisfied with the hardness of your erection? If yes, place 1 in the box. If no, place 0 in the box. 5. Were you satisfied overall with this sexual experience? If yes, place 1 in the box. If no, place 0 in the box.

Fig. 5. The Sexual Encounter Profile (SEP) diary.

detailed review on this particular matter has been recently published (Seftel, 2003). People aged 60 years or older are commonly given the image of grandparents who are somewhat sexually inactive. However, a high percentage of men over 60 years have sexual desires and are sexually active. Aging men need more time and more direct stimulation of the penis to achieve full erection (Wespes, 2000, 2002). The risk of developing ED increases with age. Almost 1 man in 22 aged 60– 69 years without previous erection problems develops ED within 1 year (Jung & Schill, 2004). Hormone replacement therapy is only indicated if testosterone deficiency is combined with the syndrome of the ‘‘aging male’’ (Jung & Schill, 2004) and lack of other disorders that are possibly causative and could be treated. A change in arterial flow velocity with age in patients has been demonstrated as well as a statistically significant tendency for reduced peak systolic velocity with age has been shown (Chung et al., 1997). The greatest decrease is present in patients between the 3rd and 4th decade of life. Another important issue to be considered is that age has a direct effect on the penis structure (Chung et al., 1997). Using a computerized image analysis, the percentage of smooth muscle cells in patients of different age with normal erections has been measured. Under 40 years, the percentage was 46%; between 41 and 60 years it was 40% and over 60 years of age it was 35% (Wespes et al., 1998; Wespes, 2000). Age also affects other important anatomical structures, such as the tunica albuginea, by altering the collagen content. Indeed, the albuginea plays an important role during the process of erection as described above. It has an equal amount of collagen

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types I and III distributed evenly along the modulating fibers, whereas type IV collagen is scant as a result of the fact that few vessels are inside the membrane (Luangkhot et al., 1992). Changes of the smooth muscle component, elastic fibers, or collagen types provoke mechanical alterations of the aging penis, thereby reducing its elasticity and compliance. ED is often associated with other conditions that are common in aging men, the most important involves the cardiovascular system (see Section 3.3.). 3.2. Diabetes Diabetes is associated with accelerated large vessel atherosclerosis and microvascular arterial disease, autonomic neuropathy, dyslipidemia, concomitant hypertension, and endothelial dysfunction. Diabetic men are 3 times as likely to develop ED as non-diabetic men. The cause is multifactorial, but most commonly reflects endothelial dysfunction and autonomic neuropathy. In a recent prevalence and risk factors analysis for ED using the International Index of Erectile Function-5, in men aged 20– 80 years undergoing a health investigation, it was shown that 32.2% of men had ED according to the IIEF-5 and that the most important risk factor was diabetes (Ponholzer et al., 2005). In a longer time frame analysis, the overall 10-year incidence of ED has been examined in men who were 21 years of age or older, were less than 30 years of age at diagnosis of diabetes, had 10 or more years of diabetes, and were taking insulin. The sample constituted 264 subjects, 25% of which developed ED with the incidence of ED increasing with age – 0.2% in the 21 –29 years old to 48.6% in those 40 years or older – and with increasing duration of diabetes (Klein et al., 2005). A similar close relation between diabetes and ED was also shown in a similar prevalence and clinical risk factors study of ED in Japanese male diabetics. The study was based on questionnaires and enrolled 82 male diabetics and 25 male nondiabetics. The endpoint used was changed into IIEF score. IIEF scores were compared between diabetics and controls and were also analyzed in relation to clinical factors. The prevalence of ED in diabetics was 60% whereas that in the age-matched controls was 20%. IIEF score decreased with duration of diabetes—ED was found to be common in Japanese male diabetics (Yamasaki et al., 2004). There is also clinical evidence that nocturnal penile tumescence (NPT) is altered in diabetic patients, indicating that there is a change in normal physiological function in people affected by diabetes. NPT studies are commonly used in the assessment of sexual dysfunction in diabetic men but it should not be taken as evidence for irreversible sexual dysfunction. Rather, the condition of diabetes appears to result in NPT abnormalities, regardless of the adequacy of daytime sexual function. Indeed, in 10 diabetic men who reported normal daytime sexual function, a significantly diminished NPT profile was found when compared with that of an age-matched, non-diabetic, healthy control group (Nofzinger et al., 1992). ED in diabetes may be secondary to a neuropathic condition of the autonomic penile nerves. Indeed, it has been shown that there is a

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functional penile neuropathic condition of the cholinergic nerves in the corpus cavernosum of diabetic impotent patients that may be responsible for the ED (Blanco et al., 1990). This relationship between autonomic neuropathy and impotence in diabetes was studied in human tissue obtained during implantation of a penile prosthesis in 19 impotent diabetic and 15 non-diabetic patients. The functional status of penile cholinergic nerves was assessed by evaluating acetylcholine accumulation, synthesis, and release by using tritiated choline. Tritiated choline accumulation and tritiated acetylcholine synthesis and release were significantly reduced in the corporeal tissue from diabetic patients compared to that from non-diabetic patients, and no differences were found between insulin-dependent and non-insulin-dependent diabetic patients (Saenz de Tejada et al., 1990). Molecular studies on human penile tissues have also shown that advanced glycation end (AGE) product formation contributes to ED by quenching nitric oxide. AGE products were found to be elevated in the penile tissue, but not the serum, of diabetic patients compared with that of non-diabetic patients. AGE products had higher levels in diabetic tunica and corpus cavernosum of diabetic patients, suggesting a tissue-specific effect of the AGEs (Seftel et al., 1997). Using tissue isolated form diabetic patients, it has also been shown through molecular studies that ED in diabetic patients is also linked to down-regulation of TGFh protein expression and the absence of the proto-oncogene Bcl-2 in the cavernosal nerve coupled to an up-regulation of p53 and HIF1a. This latter evidence suggests the possibility of hypoxiamediated ED in diabetes via up-regulation of p53 and HIF-1a (Seftel et al., 1999). Concerning the possible role of androgen in diabetics, only a single study exists, performed in 1984. It measured the plasma levels of androgens in 47 normal male controls (20 –50 years old; mean age 38 years) and in 41 male diabetics (23 – 55 years old; mean age: 37.5 years), of which 36 showed type II and 5 type I diabetes. The study showed that while the mean basal plasma LH was unchanged, there was a decrease in testosterone levels in the diabetics accompanied by an increase in testosterone binding globulin capacity and as a consequence, the apparent free testosterone concentration was markedly decreased (Ando et al., 1984). Finally, an involvement of VIP has been also shown. Immunofluorescence histochemistry in nerves in human tissue from diabetic patients with ED suggested that impairment in the VIP-releasing innervations in penile tissue may be an important factor in the development of impotence in diabetes (Crowe et al., 1983). 3.3. Cardiovascular diseases There are several pieces of evidence in preclinical pharmacology and in clinical pharmacology that cardiovascular diseases (CVD) represent a risk factor for ED. It is now generally accepted that the prevalence of CVD, such as ED, increases with age but cardiovascular diseases by themselves are one of the major risk factor for ED. Overall, atherosclerotic vascular disease can account for almost 50% of the ED in men over 50 years of age. Heart disease and its associated risk factors, such as hypertension and low serum high density

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lipoprotein cholesterol, are significantly correlated with ED as demonstrated in the MMAS study (Feldman et al., 2000). However, at present there is increasing interest in the possibility that ED represents an early sign of the existence of CVD (Russell et al., 2004), and that diagnosis of ED in an otherwise healthy patient could be considered a indication of a more systemic endothelial and vascular dysfunction. In a study on 80 patients with New York Heart Association class III/IV congestive heart failure, it was shown that ED onset can be a predictor of the presence of CVD in 40% of such patients. Another study on 40 patients with cardiac diseases found an association between sexual dysfunction and the presence of CVD as well as a correlation between the severity of ED and the number of coronary arteries with extensive atherosclerosis (Greenstein et al., 1997). The association between ED and acute myocardial infarction among men was examined in the Integrated Healthcare Information Services National Managed Care Benchmark Database (IHCIS). The IHCIS is a fully de-identified compliant database and includes complete medical history for more than 17 million in managed care; data from more than 30 U.S. health plans, covering 7 census regions; and patient demographics, including morbidity, age, and gender. A total of 12,825 ED patients and an equal number of male patients without ED were included in this retrospective cohort study. Logistic regression analyses were performed to assess the adjusted risk of acute myocardial infarction, accounting for the risk factors of age (at ED diagnosis), smoking, obesity, and medications including angiotensin-converting enzyme (ACE) inhibitors, beta blockers, and statins. The cohort of men with ED was observed to have a 2-fold increase in the risk for acute myocardial infarction after adjusting for the above risk factors. Some evidence of a possible trend toward increased risk was detected with age group. After controlling for the aforementioned co-variates and compared to men 30– 39 years of age, it was noted that the risk of developing acute myocardial infarction was 3.8-fold increased in 40- to 44-year-old patients, greater than 3-fold increased in 45- to 49-year-old patients, and 4-fold increased in 50- to 55-year-old patients (Blumentals et al., 2004). Furthermore, the IHCIS database consented to study the association between ED and peripheral vascular disease. The logistic regression analyses performed to assess the adjusted risk of peripheral vascular disease, accounting for the same risk factors as above, revealed that the cohort of men with ED had a 75% increased risk for peripheral vascular disease. Again, some evidence of a possible trend towards increased risk with age was detected. Patients aged 40 –44 years were 2.1 times more likely to develop peripheral vascular disease, 45- to 49-year-old men were also more than twice as likely to have peripheral vascular disease, and 50- to 55-yearold patients had a 3-fold increased risk of developing peripheral vascular disease. These studies clearly suggest that the risk of ED becomes more pronounced with increasing age, indicating the need to monitor ED patients who may not necessarily present with cardiovascular symptoms and that ED may serve as a marker for peripheral vascular disease (Blumentals et al., 2003). The Rancho Bernardo Study

examined whether common coronary heart disease (CHD) risk factors, measured in midlife, predict ED 25 years later. In this prospective study of community-dwelling men age 30– 69 years, 7 classic CHD risk factors (age, smoking, hypertension, diabetes, hypercholesterolemia, hypertriglyceridemia, and obesity) were assessed from 1972 to 1974. In 1998, after an average follow-up of 25 years, surviving male participants were asked to complete the International Index of Erectile Function, which allows stratification of ED into 5 groups. At the baseline there were 1810 men, but 866 died prior to follow up. As a result, 944 were contacted and 641 responded. A total of 45% of the population had mild, moderate, or severe ED, 22% did not have ED, and 31% were not active sexually. Respondents had more favorable levels of all heart disease risk factors at baseline than non-respondents. Blood pressure and fasting blood glucose were not significantly associated with ED but this finding of the study was most likely due to selective mortality (Fung et al., 2004). However, overall, this study also demonstrated that improving CHD risk factors in midlife may decrease the risk of ED as well as CHD further suggesting that there is a link between ED and cardiovascular diseases and that ED could be regarded as an ‘‘early’’ sign of cardiovascular risk. 3.4. Obesity Healthy lifestyle factors are associated with maintenance of erectile function in men and there are several studies that have correlated life-style with ED. In an early study conducted in 1999 by Chung et al., ED was evaluated in 325 patients that were classified into 2 groups according to body weight: < 120% and  120% of the ideal body weight. In this study, several erectile capacities were compared and a penile duplex ultrasonography analysis performed. In patients with obesity, there was a statistically significant decrease in the quality of residual erectile function measured as penile rigidity. Interestingly, 43% of the obese patients also displayed a significantly increased prevalence of vascular risk factors versus 30% in the non-obese, and 62% of the obese patients displayed a significant vascular impairment as assessed by duplex ultrasound versus 42% in the non-obese patients. However, when only the patients without any vascular risk factors were analyzed, no significant difference between the 2 groups was noted in the quality of residual erectile function and in the prevalence of penile vascular impairments ( P > 0.05), suggesting that obesity in itself does not seem to be an underlying factor, but does impose a risk to vasculogenic impotence by promoting development of chronic vascular disease (Chung et al., 1999). Recently, a clinical study was published that was set to determine the effect of weight loss and increased physical activity on erectile and endothelial functions in obese men. This was a randomized, single blind trial of 110 obese men (body mass index  30) aged 35– 55 years, without diabetes, hypertension, or hyperlipidemia, who had ED that was determined by having a score of 21 or less on the International Index of Erectile Function (IIEF). The 55 men randomly assigned to the intervention group received detailed advice about how to achieve a loss of 10% or more in their total body

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weight by reducing caloric intake and increasing their level of physical activity. The 55 men in the control group were given general information about healthy food choices and exercise. The study specifically monitored erectile function score, levels of cholesterol and triglycerides, circulating levels of interleukin 6, interleukin 8, and C-reactive protein, and endothelial function as assessed by vascular responses to l-arginine. After 2 years, body mass index decreased significantly more in the intervention group than in the control group, as did serum concentrations of interleukin 6 and C-reactive protein. The level of physical activity also increased significantly more in the intervention group. ED, measured as IIEF score, significantly improved in the intervention group by ¨ 20%, while it remained virtually unchanged in the control group. This study clearly demonstrated that lifestyle changes are associated with improvement in sexual function in about one third of obese men with ED at baseline (Esposito et al., 2004). In another large study, the incidence of ED in a population-based sample during a 5-year follow-up was estimated. The target population comprised all men aged 50, 60, or 70 years residing in the city of Tampere or 11 surrounding municipalities in Finland at the start of follow-up. A questionnaire was mailed to 3143 men in 1994 and to 2864 in 1999. The follow-up sample consisted of 1442 men who responded to both baseline and follow-up questionnaires. In this study, no differences were found in the incidence of ED according to the level of education, marital status, urban/rural place of residence, and amounts of alcohol and coffee consumption while obesity and current smoking significantly increased the incidence of ED (Shiri et al., 2004). In conclusion, obesity and lifestyle have an influence on the likelihood of developing ED. This is clearly shown in the studies reported here and also in other clinical studies where obesity was not the main endpoint. 3.5. Smoking Smoking is a typical risk factor for cardiovascular diseases. However, its incidence in ED has been also clinically evaluated. In a study based on the Massachusetts Male Aging Study, men with an age ranging between 40 and 70 selected from state census lists were interviewed in 1987 –1989 and reinterviewed in 1995– 1997. Analysis was restricted to 513 men with no ED at baseline and no diabetes, heart disease, or related medications at either time. Cigarette smoking at baseline almost doubled the probability of moderate or complete ED at follow-up. Cigar smoking and passive exposure to cigarette smoke in this study was also shown to significantly predict the incidence of ED (Feldman et al., 2000). Another issue that has been clinically addressed is the incidence of ED associated with cigarette smoking among middle-aged men. This was assessed from a secondary analysis of a cross-sectional survey of 4462 U.S. Army Vietnam era veterans aged 31– 49 years. The main outcome measurement was the odds ratio for reported impotence, which was calculated by comparing current smokers with non-smokers after controlling appropriately for multiple confounding factors. The study sample consisted of 1162 never smokers, 1292

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former smokers, and 2008 current smokers. The prevalence of ED found was 2.2% among never smokers, 2.0% among former smokers, and 3.7% among current smokers. The association between smoking and ED held even after adjustments were made for confounding factors, including vascular disease, psychiatric disease, hormonal factors, substance abuse, marital status, race, and age, suggesting that smoking could be an independent risk factor for ED (Mannino et al., 1994). Cigarette smoking has also been shown to be an independent risk factor in the development of atherosclerotic lesions in the internal pudendal and common penile arteries of young men with ED. This was shown in a study that evaluated arteriograms of young men with ED referred for selective pudendal angiography and found a positive correlation between smoking and ED. This study was set to evaluate the relationship between cigarette smoking and atherosclerosis of the hypogastric-cavernous arterial bed. Those patients with hemodynamically significant atherosclerosis had smoked more years than had patients without arterial disease. These differences were statistically significant for the common penile artery and the dorsal artery. Cigarette smoking was also independently associated with atherosclerosis in the internal pudendal artery when data were adjusted for age, trauma history, hypertension, and diabetes. Another analysis investigated the potential interactive effects of cigarette smoking and pelvic or perineal trauma. A significantly higher incidence of cavernous artery atherosclerosis was found among smokers (33 patients) with a history of chronic perineal trauma compared to non-smokers (25 patients) with a similar history (Rosen et al., 1991) Thus, there are strong parallels and shared risks among smoking, coronary artery disease, atherosclerosis, and ED. Clinical studies provide strong indirect evidence that smoking may affect penile erection by the impairment of endotheliumdependent smooth muscle relaxation and risk factors such as coronary artery disease and hypertension appear to be amplified by cigarette smoking. Smoking may increase the likelihood of moderate or complete ED by 2-fold (Mirone et al., 2002). Thus, it is important to point out that virtually every part of the body, including the urological system, is adversely affected by smoking. Smoking is a powerful predictor of ED; however, cessation may restore normal function. Cigarette smoke also exerts adverse effects on sperm motility and count (Mikhailidis et al., 1998). The Massachusetts Male Aging Study (MMAS) demonstrated that cigarette smoking amplifies the risk of ED associated with other risk factors such as hypertension, diabetes, dyslipidemia, and aging. At the molecular level, several changes associated with smoking have been documented in man. In particular, it has been shown that smoking causes an impaired penile flow. Cigarette smoking also interferes with the potency of intracavernous papaverine and PGE1, clearly indicating that smoking reduces not only endothelial function but also smooth muscle response (Ledda, 2000). The association between smoking and ED has also been evaluated in larger studies. In a cohort of 2115 Caucasian men aged 40– 79 years randomly selected from Olmsted County, Minnesota, smoking status was assessed by questionnaire. Of the 1329

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men with a regular sexual partner, 173 were current smokers, 836 had previously smoked, and 203 reported ED. Compared with former and never smokers, current smokers in their forties had the greatest relative odds of ED, when compared with men in their fifties, sixties, and seventies. Compared with men who never smoked, men who smoked at some time had a greater probability of ED and with the age-adjusted odds ratio it was even possible to define a dose-response. This clinical study has good parallelism with early animal studies on smoking and ED. In a study performed in healthy adult mongrel dogs, for example, bipolar cuff electrodes were implanted around the cavernous nerves. After threshold stimulation parameters for penile erection were established, dogs were exposed to cigarette smoke collected in a 60-mL syringe and released slowly near the dog’s mouth, to be inhaled by natural breathing. Five of the 6 dogs did not achieve full erection after inhalation of smoke from 2 to 3 cigarettes. Some decrease of flow through the internal pudendal artery occurred and the venous restriction ability was almost completely abolished by smoking. Similarly, when nicotine was injected intravenously into 2 additional dogs, the same phenomenon was observed (Juenemann et al., 1987). In conclusion, smoking can be regarded in man as almost an independent risk factor for ED as it is for cardiovascular diseases. 4. The mediators The penis is kept in a flaccid state by a predominance of aadrenergic tone. Indeed, tonic noradrenaline released from the sympathetic nerves contracts penile arterial and corporal smooth muscle through activation of post-junctional a1receptors mainly by increasing intracellular calcium. Noradrenaline also inhibits vasodilatory neurotransmitter released by pre-junctional a2-receptors. Other mediators that have been implicated in modulating contractile tone are endothelins, angiotensin among many others. Recently, many studies have focused on the role played by activation of the Rho-kinase pathway in mediating the contractile Fmessage_ in penile tissue. Penile vasodilatation is mainly mediated by the l-arginine – NO pathway through activation of guanylyl cyclase in the smooth muscle component, followed by an increase in cGMP that in turn lowers intracellular calcium. Acetylcholine, vasoactive intestinal peptide, peptides in sensory nerves, and prostanoids have also been shown to be involved in penile vasodilatation. 4.1. Contraction Here follows a review of all the major contractile factors involved in the tone responsible for the flaccid state. Particular attention has been given to those factors for which studies on human tissue and clinical data are available. Other factors are not discussed either because human data are not available or because there are very few and/or there is no convincing evidence for a role in man. Data from preclinical studies on animals are reported only in the section on the Rho-kinase pathway because this pathway at the moment represents the

most promising and intriguing alternative therapeutic target in developing drugs for ED not based on phosphodiesterase-5 inhibition. 4.1.1. The adrenergic system Adrenergic tone is mainly responsible for keeping the penis in the flaccid state. This view is supported by the findings that in the human corpus cavernosum, the density of a-adrenoreceptors are almost 10 times higher than that of h (Levin & Wein, 1980). In human corpus cavernosum, the presence of a1and a2-receptors, with a predominance of a1-receptors, has been shown (Traish et al., 1995a, 1995a, 1997a, 1997b, Goepel et al., 1999). The adrenoreceptor subtypes a1A, a1B, and a1D have been found in human corpus cavernosum (Traish et al., 1995a, 2000; Dausse et al., 1998), with a predominance of a1A, a1B, and a1D receptor protein (Traish et al., 1995a, 1997b; Goepel et al., 1999). Regarding a1B receptors, the presence of a homogeneous population of a2A adrenoreceptors in human tissue has been shown (Goepel et al., 1999; Traish et al., 2000). Using cultured human corpus cavernosum smooth muscle cells with the stimuli PGE1 and forskolin, it has been shown that there is an increase in a2A adrenergic receptor expression determined by RNase protection assays. This effect was coupled to a marked increase in cAMP production. The presence of functional a2-receptors in human corpus cavernosum has been also recently confirmed by others using the agent yohimbine. However, the effect of yohimbine in this study has not been shown to be purely dependent upon a2-receptors because yohimbine also prevented contraction induced by ET-1 in a manner unrelated to ET receptors (Filippi et al., 2002). Recently, the presence of h3-adrenoreceptors in human corpus cavernosum has also been shown, and that this subtype is mainly localized in smooth muscle cells (Cirino et al., 2003). Interestingly, activation of this receptor in human corpus cavernosum leads to a cGMP-dependent but NO-independent vasorelaxation selectively blocked by a specific h3-receptor antagonist. In the same study, it was also demonstrated that the human corpus cavernosum exhibits basal h3-receptor-mediated vasorelaxant tone and that h3-receptor activity is linked to inhibition of the Rho-kinase pathway. There are several clinical studies that have been performed in order to give a proof of concept of the possible use of an a-antagonist in the treatment of ED. While some of these studies are obsolete from the therapeutic point of view, they are crucial in order to understand the role played by a receptors in human erectile function. One of these clinical studies was a single center, observer-blinded, randomized, placebo-controlled, extended period Latin-Square crossover design conducted in 24 male patients of mean age 44 years with ED of no established organic cause. The drug tested was a selective a1A antagonist, namely, Ro70-0004, administered at a dose of 5 mg orally (Choppin et al., 2001). The primary efficacy endpoint was the duration of rigidity > 60% at the base of the penis measured between 0.5 and 2.5 h post-dose. Sildenafil (50 mg) was used as a positive control. The antagonist did not improve erectile function when compared to placebo as opposite to sildenafil.

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However, clinical studies have also been performed on wellknown non-selective a1-receptor inhibitors such as phentolamine, giving positive results. In particular, phentolamine, at doses of 40 or 80 mg that produce mean plasma phentolamine concentrations sufficient to occupy a1- and a2-adrenergic receptors in erectile tissue, was shown to be active (Goldstein, 2000). In this placebo-controlled pivotal phase III clinical trial, the endpoint was the mean change in the erectile function domain of the International Index of Erectile Function score. The score from the initial screening to the end of treatment was significantly higher following the use of phentolamine when compared to placebo. Fifty-five percent of patients at doses of 40 mg and 55% of patients taking the 80 mg dose, respectively, were able to achieve vaginal penetration, with 51% and 53% achieving penetration on 75% of attempts, respectively. There are no recent clinical studies on a2-receptors, and looking over the past decade we found only 2 studies addressing the possible therapeutic use of a2-receptor blockade in a clinical trial. In a study published in 1994, the effects of an a2-adrenoceptor antagonist, RS 15385, on sleep and nocturnal penile tumescence were assessed by intravenous infusion of the drug at 2 doses (high and low) through the night in 12 young normal volunteers and 24 men with ED. The drug reduced total sleep time in the younger normal volunteers with the high dose only and in the dysfunctional men with both doses. In the volunteers, there was a curvilinear dose – response effect on nocturnal penile tumescence. The lower dose of the drug only modestly increased erectile response, particularly during non-REM sleep, whereas the higher dose reduced erectile response, most noticeably during REM. The only positive effect found in this study was that the drug at the higher dose tested caused an increase in spontaneous erections after lights out before sleep onset (Bancroft et al., 1995). In a parallel study, the effect of the same a2-adrenoceptor antagonist on erectile function in 24 men with probable psychogenic ED was assessed. The drug was given in 2 doses, together with placebo, by intravenous infusion in a balanced cross over design. Once plasma levels were established, erectile, subjective, and hemodynamic responses to erotic fantasy and films were measured. Subjects were divided into 2 age groups, ‘‘younger’’ (< 45 years) and ‘‘older’’ (> 45 years). The final outcome was the presence of a significant though modest increase in the duration of erectile response with the high dose of the drug, but only in the younger men. Thus, there is not a big contribution of a2-receptors to erectile function and the only open unresolved issue is whether in younger men with psychogenic erectile failure an a2 tone might be responsible of the inhibition of the general arousal responses to erotic stimuli (Munoz et al., 1994). 4.1.2. Endothelin Endothelins (ETs) are a family of endogenous peptides, mainly secreted by endothelial cells, which exert a potent vasoconstrictor and pressor activity, acting through 2 classes of receptors named ETA and ETB. ETs were originally isolated from porcine aortic endothelium (Yanagisawa et al., 1988; Inoue et al., 1989), but subsequent studies revealed that ETs are

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synthesized and their receptors are present in a variety of tissues, where they play important physiological and pathophysiological roles. Endothelins have been suggested to contribute to the maintenance of corporal smooth muscle tone. Indeed, endothelial cells from the human corpus cavernosum, but not nonendothelial cells, were found to express ET-1 mRNA (Saenz de Tejada et al., 1991). ET-like immunoreactivity was observed in the sinusoidal and also in cavernous smooth muscle (Saenz de Tejada et al., 1991). Autoradiography studies have shown that binding sites for ET-1 are present both in the vasculature and trabecular tissue of the human corpus cavernosum (Holmquist et al., 1990, 1992). Both ETA and ETB receptors have been found in human corpus cavernosum smooth muscle membranes (Christ, 1995). ET-1 induces potent contractions in vitro that are slowly developing and long-lasting in corpus cavernosum, cavernous artery, deep dorsal vein, and penile circumflex veins, demonstrating that different penile smooth muscle cells respond equally to ET-1 (Becker et al., 2000). ET-2 and ET-3 also causes human corpus cavernosum to contract, although they are less potent than ET-1 (Saenz de Tejada et al., 1991). Different mechanisms have been shown to be involved in ET1-induced contractions. In particular, transmembrane calcium flux has been shown to be due to voltage-dependent and/or receptor-operated calcium channels, whereas calcium mobilization involves inositol 1,4,5-trisphosphate-sensitive intracellular calcium stores (Holmquist et al., 1990, 1992). The possible role played by ET-1 in the pathophysiology of erection has been addressed in clinical studies. Christ (1995) found no detectable age- or diabetes-related changes in contractile effects in human corpus cavernosum tissue. Similarly, Francavilla et al. (1997) found no differences in the plasma concentrations of ET-1 in diabetic and nondiabetic patients with ED. Furthermore, the concentrations of ET-1 in cavernous blood were not modified during penile erection induced by intracavernous PGE1 injection. In another study, systemic and cavernosal plasma levels of endothelin (1– 21) were measured during different penile conditions in healthy males and patients with ED. In this study, ET-1 and ET-2 plasma levels were measured in systemic and cavernosal blood taken from healthy males and patients with ED during different penile conditions, such as flaccidity, tumescence/rigidity, and detumescence. The study groups comprised 33 healthy adult males and 25 patients that were exposed to visual and tactile erotic stimuli in order to elicit penile tumescence and, in the group of healthy volunteers, rigidity. In the healthy males, no changes in ET-1 or ET-2 levels were observed in the systemic and cavernosal blood during penile tumescence, rigidity, and detumescence. In the ED patients, mean plasma ET-1 and ET-2 levels during penile flaccidity and detumescence were found to be higher in the systemic circulation than in the cavernosal blood. No differences in the plasma courses of ET-1 and ET-2 were found between patients with organogenic or psychogenic ED etiology. During detumescence, the mean ET level was lower in the cavernosal blood taken from the patients than in the samples obtained from the healthy males. Our study

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revealed a difference in the profiles of ET-1 and ET-2 in the cavernosal blood of healthy subjects and patients with ED. However, this difference does not appear to be of physiological significance, implying that in man, ET-1 may have only relative importance in the control of penile flaccidity and thus in the pathophysiology of ED (Becker et al., 2001a). Similarly, in another study, ET levels were measured in 33 patients with arteriogenic impotence before and after administration of papaverine. Intracavernosal levels of endothelin were not altered either in the flaccid or papaverine-induced erectile status (Kadioglu et al., 1998). Even if the accumulated information suggests that ETs may have a role in the mechanisms of flaccidity and detumescence, their exact role in penile physiology and pathophysiology remains to be established. ETs may function not only as a long-term regulator of corporal smooth muscle tone, but also as a modulator of the contractile effect of other agents, for example, noradrenaline (Holmquist et al., 1990; Christ, 1995; Kim et al., 1996). It was shown recently that ETA receptors are expressed by both endothelial and muscular cells isolated from human corpus cavernosum and that penile endothelial cells only express ETB receptors, whose function is enhanced during experimental hypoxia (Filippi et al., 2003). In conclusion, although the mechanistic studies performed both in vivo and in vitro in experimental animals suggest a role for ET-1 in flaccidity or detumescence, studies on human tissue do not support this view. The fact that ET-1 may not play this key role in human tissue is further supported by the only clinical study so far available in the literature. The ETA receptor antagonist BMS193884, at an oral dose of 100 mg/kg, was tested versus placebo in 53 men diagnosed with mild-to-moderate ED in a double-blind fashion. Although the drug was well tolerated, BMS-193884 did not significantly improve erectile function during office visits or home use when compared to placebo. In the same study, the antagonist worked in rabbits when tested both in vitro and in vivo (Kim et al., 2002). Thus, a disparity exists between laboratory and clinical studies, suggesting that differences between species should not be underestimated in defining the pathophysiology of erectile function. 4.1.3. Angiotensin and angiotensin-converting enzyme Angiotensin II (AngII) is an octapeptide hormone that is the active component of the renin – angiotensin system. It regulates blood pressure, plasma volume via aldosterone-regulated sodium excretion, sympathetic nervous activity, and thirst responses. In mammalian cells, AngII mediates its effects via at least 2 high-affinity plasma membrane receptors, AT1 and AT2, which have been cloned and pharmacologically characterized (for a review, see Touyz & Schiffrin, 2000). Human corpus cavernosum produces and secretes physiologically relevant amounts of angiotensin II (Kifor et al., 1999), and in vitro AngII causes a dose-dependent contraction of human corpus cavernosum strips. In man, it has been shown that AngII plays an important role in regulating the flaccid state and the detumescence of the penis. In an elegant clinical study, the functional effect of AngII on different functional conditions of penile erectile tissue was evaluated by measuring AngII levels

in cavernous and peripheral blood samples taken from cubital vein of healthy volunteers. During the flaccid and the detumescence phase, there is an increase in AngII levels. Indeed, in this study, the mean AngII level in the cavernous blood was about 30% higher than that in the systemic circulation, strongly supporting the hypothesis that AngII is involved in flaccidity and detumescence (Becker et al., 2001b). Involvement of AngII has been also shown in pathological conditions. In another clinical study, 34 healthy adult men and 48 patients with ED of either organogenic or psychogenic etiology were exposed to visual and tactile erotic stimuli to elicit penile tumescence versus a group of healthy subjects where stimulation caused rigidity. Whole blood was simultaneously aspirated from the corpus cavernosum and the cubital vein during the different functional conditions of the penis. In healthy men, the AngII levels in the cavernous plasma increased by 28%. A similar but weaker increase was found in peripheral plasma. During penile flaccidity, the AngII levels in the systemic and cavernous blood were higher in the group of organogenic patients than in the control group. This study also suggest that AngII might be involved in the initiation of penile detumescence in men and that AngII plasma levels could be an useful tool in the diagnosis of organogenic ED (Becker et al., 2001c). The angiotensin-converting enzyme (ACE) may also be involved in the pathogenesis of ED. ACE levels were measured in the flaccid penis cavernosal blood of ED patients and in cubital blood of ED patients and controls. A total of 36 ED patients, made up of 12 organogenic diabetic patients, 12 organogenic non-diabetic patients and 12 psychogenic patients, and 12 healthy adult male controls were enrolled in this study. In the diabetic and non-diabetic ED patients, systemic ACE activity was enhanced and in diabetic ED patients, cavernosal ACE activity levels were also significantly elevated. In addition, this study demonstrated that systemic NO levels negatively correlate with ACE activity in diabetic patients (Hamed et al., 2003). The D polymorphism of angiotensin converting enzyme gene has been found to be associated with various diseases. On the other hand, interpretation of the data on the association of DD genotype with various diseases is controversial due to methodological and technical variations in detection of the polymorphisms. The various studies addressing the relationship between this polymorphism and ED have produced conflicting results. A German group monitored the response to sildenafil in 113 men with ED, according to the patients’ diaries, and compared their ACEI/D and NOS3 genotypes with 108 healthy male blood donors, demonstrating that patients with elevated ACE serum concentrations, as associated with the D allele of the ACE I/D polymorphism, are less likely to respond to sildenafil (Eisenhardt et al., 2003). On the other hand, in a frequency analysis of 894 G/T (Glu298Asp) eNOS and ACE I/D polymorphisms in 53 Mexican ED patients and 62 age-matched controls, it was demonstrated by multiple logistic regression analysis that the Glu298Asp polymorphism was an independent factor for ED, but no association was found between ACE I/D polymorphism and ED in the population studied (Rosas-Vargas et al., 2004). In a further study, the analysis of ED patients and their

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genotypes regarding the GNB3 C825T, the ACE I/D, and the NOS3 G894T polymorphisms did not show evidence of influence of these genotypes on susceptibility to ED development. At the same time, a significant variation in drug response to sildenafil that was dependent on ACE I/D polymorphism genotype was demonstrated. Only 50% of ACE D allele carriers showed a positive response to sildenafil, in contrast to men that had genotype II in the ACE I/D polymorphism, who had a response rate of 75% (Eisenhardt & Siffert, 2003). However, the methodology used for determination of the polymorphism can produce variability in the results. Indeed, in a study where the possible association between the DD genotype and ED in the Korean population was investigated, the frequency of ACE genotypes detected using a multiplexed PCR method was compared to that produced by a conventional PCR method. The results suggested that the conventional PCR method for ACE gene polymorphism detection requires careful control and may need repeated testing to verify the insertion deletion (ID) heterozygotes, and that a multiplexed PCR method can markedly increase the detection rate of the I allele in ID heterozygotes. The final outcome of this study was the lack of association between I/D polymorphism and ED patients in the Korean population studied (Kim et al., 2001). Clinical studies have also tried to evaluate the role of ACE and AngII through the use of the selective AngII blockers commercially available. In particular, 2 studies exist showing a beneficial effect of AngII receptor blockers on ED. In an open and prospective study, the effect of the AngII receptor blocker valsartan on sexual function in hypertensive males was studied. Patients who were either newly treated, or who were switched from other treatment regimens, received valsartan 80 –160 mg/ day. The patients’ sexual function was assessed before and after 6 months of valsartan treatment using the International Index of Erectile Function (IIEF). At baseline, 75.4% of the total group of 3502 patients investigated and 65.0% of the subgroup of patients without previous antihypertensive treatment (n = 952) could be diagnosed as having ED according to the IIEF. Valsartan therapy markedly reduced ED in these groups to 53% and 45%, respectively. The amelioration of the ED was associated with highly significant improvements in orgasmic function, sexual desire, intercourse , and overall satisfaction both in the total and previously untreated groups (Dusing, 2003). A similar study evaluated the influence of a 12-week therapy with losartan in 82 hypertensive subjects with (n = 82) and without (n = 82) a diagnosis of ED using a selfadministered questionnaire. Losartan (50 – 100 mg/day) treatment improved sexual satisfaction from an initial of 7.3% to 58.5%. However, at the completion of the 12-week losartan regimen, only 11.8% of the treated subjects reported in improvement in sexual function. Improvement of quality of life was demonstrated in 73.7% of subjects medicated with losartan, while 25.5% reported no changes, and only 0.8% felt worse. In the group without sexual dysfunction, losartan had a non-significant effect on sexual function (Llisterri et al., 2001). In conclusion, from clinical studies it appears clear that there is an involvement of ACE and AngII in the pathophysiology of

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ED. Indeed, there are clear data that show involvement of AngII in flaccidity and detumescence as well as the pharmacological clinical evidence from the use of selective AngII receptor blockers that AngII is involved in organogenic and psychogenic ED. 4.1.4. The Rho-kinase pathway The flaccid state of the penis is driven by the release of several contractile mediators. The most important in modulating the peripheral vascular tone in the man has already been described in the previous sections. Antagonism at the receptor level should lead to a decrease in myosin regulatory light chain phosphorylation and to relaxation, by decreasing cytoplasmic calcium concentration and/or inhibiting calcium sensitization. Calcium sensitization of smooth muscle cells takes place through inhibition of the smooth muscle myosin light chain phosphatase (MLCP) that dephosphorylates the regulatory myosin light chain. Contractions induced at constant intracellular calcium concentration by certain agonists are due to inhibition of the myosin phosphatase, whereas the inhibitory signal for calcium sensitization is communicated by RhoA to Rho-kinase. The latter subsequently phosphorylates a regulatory subunit that inhibits the catalytic activity of MLCP, eventually resulting in increased regulatory myosin light chain phosphorylation, contraction, and cell motility. Calcium release can be dissociated by calcium sensitization but inositol triphosphate does not sensitize smooth muscle, excluding phospholipase C products as major calcium-sensitizing effectors. Indeed, some very potent calcium sensitizing agonists, such as the thromboxane analogue U46619, cause little or no release of intracellular calcium, less than would be expected by an activated G-protein-coupled receptor complex. RhoA is a monomeric G-protein that is active when it contains bound GTP and inactive when the bound nucleotide is GDP. The calcium-sensitizing effector of RhoA-GTP is a serine/threonine kinase generically referred to as Rho-kinase, which contains a Rho-binding domain. Activated Rho-kinase phosphorylates the regulatory subunit of MLCP and inhibits phosphatase activity. It is intuitive that the calcium-sensitizing Rho-A/Rho-kinase pathway may play a synergistic role in cavernosal vasoconstriction to maintain the flaccid state. This role has been demonstrated in experimental animals using the Rho-kinase inhibitor Y-27632. Rho-kinase inhibition stimulates rat penile erection independently of nitric oxide in vivo and causes relaxation in vitro (Chitaley et al., 2001). This early evidence has been followed by 2 key studies showing that (i) RhoA mediates calcium sensitization in erectile function and (ii) RhoA expression is controlled by nitric oxide in a cGMPdependent manner. In particular, it has been shown that human corpus cavernosum contains RhoA, whereas Ca+ 2 sensitization of permeabilized human corpus cavernosum has been directly demonstrated with identification of a highly expressed molecular component of this pathway. Calcium sensitization of force induced by endothelin or GTPgS is significantly greater and associated with a 17-fold higher RhoA content in human corpus cavernosum than in rabbit ileum smooth muscle. Pulldown assays showed that the high RhoA content of corpus

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cavernosum was available for activation by GTPgammaS. Human corpus cavernosum expresses the phosphatase inhibitor CPI-17, the myosin phosphatase regulatory (MYPT-1) and catalytic (PP1delta) subunits, and 2 isoforms of Rho-kinase. Thus, it appears that the high expression of RhoA contributes, through RhoA-mediated Ca+ 2 sensitization, to the flaccid state of the corpus cavernosum (Wang et al., 2002). In the second molecular study, the effect of NO signaling on RhoA expression in arterial smooth muscle cells was analyzed both in vitro and in vivo. In human artery smooth muscle cells, sodium nitroprusside, or 8-(2-chlorophenylthio)-cGMP induced a rise in RhoA mRNA and protein expression that was inhibited by the cGMP-dependent protein kinase (PKG) inhibitor (R(p)-8-bromo-beta-phenyl-1,N(2)-ethenoguanosine 3V:5V-phosphorothioate. The NO/PKG-dependent stimulation of RhoA expression involved both an increase in RhoA protein stability and stimulation of RhoA gene transcription. Cloning and functional analysis of the human RhoA promoter showed that the effect of NO/PKG involved phosphorylation of ATF-1 and subsequent binding to the cAMP response element. Chronic inhibition of NO synthesis, obtained by treating rats in vivo with l-NAME, strongly decreased RhoA mRNA and protein expression in aorta and pulmonary artery and this was associated with inhibition of RhoA-mediated Ca+ 2 sensitization. Such data, taken together with the finding that sildenafil prevented these effects, demonstrates that NO/PKG signaling positively controls RhoA expression and suggests that basal release of NO is necessary to maintain RhoA expression and RhoA-dependent functions in vascular smooth muscle cells (Sauzeau et al., 2003). There are several studies that have addressed the possible role played by this pathway in ED associated with diabetes. In a recent study, co-localization of Rho-kinase and eNOS protein in the endothelium of the corpus cavernosum of streptozotocin diabetic rats was shown. In addition, eNOS protein expression, cavernosal constitutive NOS activity, and cGMP levels were reduced in the STZ diabetic penis. A functional role for the RhoA/Rho-kinase in the penis was demonstrated in this study by using an adeno-associated virus encoding the dominantnegative RhoA mutant AAVTCMV19NRhoA. STZ diabetic rats transfected with the RhoA mutant AAVCMVT19NRhoA had a reduction in RhoA/Rho-kinase and MYPT-1 phosphorylation while cavernosal eNOS protein, constitutive NOS activity, and cGMP levels were restored to the levels found in the control rats. These data suggest a possible downregulation of penile eNOS in diabetes mediated by activation of the RhoA/Rho-kinase pathway. In other words, inhibition of the RhoA/Rho-kinase improves eNOS protein content and activity, thus restoring erectile function in diabetes (Bivalacqua et al., 2004). Other studies have suggested a key role for the Rho-kinase pathway in diabetes but all of them have been performed on diabetic mice and rabbits. Thus, so far, there are no clinical studies that have directly addressed the role of the Rho-kinase in ED. However, there are clinical studies that have addressed the role of this pathway in patients affected with cardiovascular disease, whose link with ED is well established. Through the evaluation of the forearm blood flow in healthy

young male smokers and non-smokers, it has been shown that the vasodilatory effect of fasudil, a Rho-kinase inhibitor, was significantly greater in smokers than in non-smokers, suggesting that smoking activates Rho-kinase in forearm vascular smooth muscle cells in vivo. Interestingly, smoking did not alter the vasodilatory effect induced by exogenous nitric oxide in forearm (Noma et al., 2003). Fasudil has also been shown to ameliorate myocardial ischemia induced by intracoronary acetylcholine in patients with angina and normal epicardial coronaries (Mohri et al., 2003). This effect of fasudil was also confirmed in a multicenter phase II study in patients with stable effort angina (Shimokawa et al., 2002) and in the prevention of ACh-induced coronary artery spasm and resultant myocardial ischemia in patients with vasospastic angina (Masumoto et al., 2002). In conclusion, the Rho-kinase pathway surely plays an important role in the contractile flaccid tone of the human penis, and if the parallels between ED and cardiovascular disease continue to be a good predictor, the Rho-kinase pathway surely will represent an alternative therapeutic target for the treatment of ED. 4.2. Relaxation The pathways involved in relaxation have been more deeply investigated in the past 10 years, with the available data clearly pointing to a key role for the l-arginine –NO pathway and cGMP. Indeed, the inhibitors of phosphodieaterase-5 represent the first class of orally available drug for the treatment of ED. In the following section, the role of NO and other mediators in the modulation of the relaxant response of the human corpus cavernosum will be reviewed. 4.2.1. l-Arginine– nitric oxide pathway and guanylyl cyclase/cGMP pathway There are several reviews addressing the key role of NO in ED (for an updated review, see Toda et al., 2005). Here we simply summarize the role played by the l-arginine/NO pathway in the man. An important role for NO in the relaxation of corpus cavernosum smooth muscle and vasculature is widely accepted (Burnett, 1997; Simonsen et al., 2002; Toda et al., 2005). NO can be produced by the endothelium and/or the nerves innervating the corpus cavernosum. The presence of nNOS has been shown in the pelvic plexus, cavernous nerve, and adventitia of the deep cavernous arteries in men by NADPH diaphorase staining and immunoreactivity. In addition, it has been recently shown, by using confocal microscopy and double immunolabeling for nNOS and the vesicular acetylcholine transporter, that nNOS immunoreactivity coexists in the majority of nerve terminals surrounding human penile arteries. NO has been also shown to mediate neurogenic non-cholinergic non-adrenergic (NANC) relaxation in human penile resistance arteries, in large penile arteries and in veins (Simonsen et al., 2002; Toda et al., 2005). Examination of NOS enzyme activities in specimens of potent and impotent patients has been performed by means of light and electron microscopy, using NADPH diaphorase staining and eNOS-specific and smooth muscle actin-specific immunohisto-

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chemistry (Bloch et al., 1998). They found a distinct expression of eNOS in cavernosal smooth muscle and in the small intracavernosal helicine arteries. No overall correlation between NOS expression and erectile function was observed. In human penile cavernosal smooth muscle cells in culture, Rajasekaranan et al. (1998) found mRNA expression of both eNOS and inducible NOS. Localization studies showed positive signals for NADPH diaphorase, eNOS, and calmodulin, and electron microscopic evaluation confirmed the localization of eNOS to the cytoplasm and small vesicles in the cells. Stanarius et al. (1999), using electron microscopy and immunohistochemistry, detected eNOS in the endothelial cells covering the cavernous spaces and in the endothelial cells of arteries branching within human erectile tissue. They found no eNOS activity in cavernous smooth muscle cells and cavernous nerves. Caveolin-1 has been shown to be an endogenous modulator of eNOS activity (Fulton et al., 2001; Gratton et al., 2004). Expression of caveolin-1 and caveolin-3 was evaluated in human corpus cavernosum in a study by Tsutsui et al. (1999). The study revealed that caveolin-1 is diffusely located within the smooth muscle of the corpus cavernosum and endothelium of the vasculature. Interestingly, caveolin-3, which binds to nNOS, was located close to NADPH-positive nerve fibers (Tsutsui et al., 1999). Functional studies also support the occurrence and importance of eNOS in human cavernous tissue (Simonsen et al., 2002; Toda et al., 2005). It is worth mentioning that in streptozotocin-induced diabetic rats, adenoviral gene transfer of endothelial nitric oxide synthase to the penis increased peak and total intracavernous pressure resulting from cavernous nerve stimulation to a level similar to control rats (Bivalacqua et al., 2003). The action of NO is explained by activation of the soluble guanylyl cyclase (sGC). The GC exists in 2 isoforms: a particulate isoform that is membrane bound and a soluble isoform that is expressed in nearly all cell types (Lucas et al., 2000). In the penis, soluble GC is probably the most important receptor for NO as a signaling molecule. The enzyme, which catalyzes the conversion of GTP into cyclic GMP, consists of 2 different subunits and contains a prosthetic heme group that mediates up to 400-fold activation by NO. Using RNA extracted from tissue samples obtained from 7 patients undergoing penile prosthetic surgery or correction of penile deviation, a molecular study on sGC expression was performed. By reverse transcriptase – polymerase chain reaction with specific primers for the subunits of NO-sensitive guanylyl cyclase, specific amplification products encoding the alpha(1), beta(1), alpha(2), and beta(2) subunits were detected as well as a novel h2 variant by alternative splicing from the same gene as the h2 subunit (Behrends et al., 2000). Functional studies on human tissue have also been performed with a specific chemical stimulator of sGC, YC-1 [3-(5-hydroxymethyl-2-furyl)-1-benzylindazole], which directly stimulates sGC in the presence of sodium nitroprusside and causes 2200-fold stimulation of human recombinant sGC (Lee et al., 2000). BAY 41-2272 (5-Cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-xylamine), a potent soluble guanylyl cyclase stimulator, powerfully relaxed human corpus cavernosum in a NO-independent manner in vitro (Baracat et al., 2003). mRNA transcripts encoding for

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guanylyl cyclase-B (the bound or particulate form), which acts as a receptor of the peptide hormone C-type natriuretic polypeptide, have been also identified in human corpus cavernosum. This finding was verified at the protein level by immunohistochemistry that demonstrated the presence of guanylyl cyclase B in human corpus cavernosum (Kuthe et al., 2003). Cyclic GMP signals via 3 main effector mechanisms in eukaryotic cells: ion channels, phosphodiesterases, and protein kinases (Lucas et al., 2000). Two different cGMP-dependent protein kinases, namely, cGKI and cGKII, have been identified in mammals. The expression of cGK I was examined in corpus cavernosum specimens from patients with and without ED (Klotz et al., 2000). In all specimens of cavernosal tissue, a distinct immunoreactivity was observed in different parts and structures, with a high expression in smooth muscle cells of vessels and in the fibromuscular stroma. No clear immunoreactivity against cGK I was found in the endothelium. There was no distinct difference in immunoreactivity and cellular distribution between potent and impotent patients. 4.2.2. Prostanoids It is well established that human corpus cavernosum tissue can synthesize prostanoids (Miller & Morgan, 1994; Porst, 1996; Minhas et al., 2000; Andersson, 2001) and that the production of prostanoids can be modulated by oxygen tension and suppressed by hypoxia (Daley et al., 1996a, 1996b). The effects of prostanoids are mediated through prostanoid receptors that are G-protein-coupled with differing transduction systems (Coleman et al., 1994; Pierce et al., 1995; Narumiya et al., 1999). The effects of prostanoids are also mediated through specific receptors. In human corpus cavernosum, cDNAs encoding representatives for DP (PGD2), EP (PGEs), FP (PGF2a), IP (PGI2), and TP (TXA2) receptors have been cloned, including several subtypes of EP receptors (Moreland et al., 1999). However, the role of all these different receptors is not well established as yet (Khan et al., 1999). PGE1 induces relaxation of human corporal smooth muscle and it is also clinically used for the diagnosis of psychogenic or organogenic ED. It has also been suggested to activate Ca+ 2-dependent potassium channels, resulting in hyperpolarization (Lee et al., 1999). Prostanoids exert their effects mainly through cAMP. Palmer et al. (1994) found that forskolin, which directly stimulates adenylate cyclase, was a potent stimulant of intracellular cAMP formation in cultured human corporal smooth muscle cells. Threshold forskolin doses were found to significantly increase the production of cAMP by PGE1, which suggested a possible synergistic effect. Traish et al. (1997a) confirmed this synergistic effect of forskolin and PGE1 in cultured human corpus cavernosum cells. They also demonstrated that the augmentation of the forskolin-induced cAMP generation by PGE1 was mediated by EP receptors and attributable to interactions at the adenylyl cyclase and Gprotein levels. Both forskolin and PGE1 elicited concentrationdependent increases in the magnitude and duration of intracorporal pressure in dogs without systemic effects. Mulhall et al. (1997) injected forskolin intracavernously to patients with ED who had failed to respond to standard

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injection therapy and found improvement of erection in 61% of the cases. These results suggest that it is possible to enhance the relaxant corporal effects of PGE1, and possibly other vasodilators, by forskolin and analogues, and it cannot be excluded that this may provide new strategies for pharmacologic treatment of ED. Another way of enhancing the effects of PGE1 may be to combine treatment with a-adrenoceptor antagonists, such as doxazosin (Kaplan et al., 1998). The functional EP receptors, EP1, EP2, and EP3, have been characterized in human corpus cavernosum tissue and in human corpus cavernosal smooth muscle cells by RNase protection assays. A functional characterization was also performed using human corpus cavernosum strips and selective EP1/EP2/EP3 receptor antagonists (Moreland et al., 2003). Interestingly, while the clinical benefit of PGE1 in ED in men is well proven, there is almost no response in other species including non-human primates. A PGE1 binding study performed using human tissue as well as corpus cavernosum tissue from rhesus and cynomolgus monkeys showed the presence of high- and low-affinity PGE1 binding sites only in human tissue (Meghdadi et al., 1999). In another study, it was shown that endogenous production of prostanoids is involved in the regulation of penile smooth muscle contractility by way of specific receptors. TP receptors mediate contraction in human corpus cavernosum strips and human penile resistance arteries, while the relaxant effects of prostanoids are mediated by EP2and/or EP4-receptors in human corpus cavernosum and by EP and IP receptors in human penile resistance arteries (Angulo et al., 2002). Injection of intracavernous PGE1 causes vasodilatation by relaxing the smooth muscles of the corpus cavernosum and by increasing the diameter of cavernous arteries, leading to erection. At therapeutic doses, intracavernous alprostadil is well tolerated. The most common adverse event, transient penile pain, occurred in around one third of patients and in 11% of injections, causing 3 – 5% of patients to withdraw from self-injection programs. Injected PGE1 is either locally metabolized or rapidly cleared from the penis into the systemic circulation, where it is extensively metabolized by the lungs. Intracavernous PGE1 was largely used before the advent of the oral inhibitors of phosphodiesterase-5 (PDE5). In this context, studies have clearly shown that repeated injection of PGE1 can be performed without any loss of activity and that treatment with intracavernous PGE1 with 150– 250 injections of 5– 20 Ag did not cause histological changes of intracavernous smooth muscle (Wespes et al., 2000). At present, PGE1 is currently used for the diagnosis of organogenic or psychogenic ED and their use in ED treatment is limited to non-responders to PDE5 inhibitor therapy. 4.2.3. Vasoactive intestinal polypeptide The first demonstration of high concentrations of vasoactive intestinal polypeptide (VIP) was obtained in 1983 in 30 surgical specimens of male external genitalia by immunocytochemistry and radioimmunoassay (Willis et al., 1983). In this original study, VIP was found exclusively in fine autonomic nerves. VIPcontaining nerves were most densely concentrated in the penis around the pudendal arteries and in the erectile tissue of the

corpus cavernosum. Considerable numbers of VIP nerve fibers were also seen in the vas deferens and epididymis (Polak et al., 1981). VIP is a potent relaxant of human corpus cavernosum, with this effect present at very low doses (0.03 nM) and blocked by VIP antiserum. Intracavernous injection of VIP (1.0 Ag) in human volunteers caused some degree of penile enlargement but no erection in 5 of the 7 subjects (Adaikan et al., 1986a, 1986b). The presence of VIP has been also shown in human circumflex vein by immunoreactivity and through functional studies. In particular, in circumflex vein pre-contracted with noradrenaline, VIP caused a maximal relaxation of 80% of at the maximal dose tested (10 6 M) (Kirkeby et al., 1992). Plasma levels of VIP were also measured in 12 patients, made up of 6 men with psychogenic impotence, 2 with induratio penis plastica, and 4 with impotence of vascular origin. The concentrations of VIP in corpus cavernosal blood during pharmacologically induced erection with PGE1 did not increase in any of the patients treated. In addition, the VIP concentrations in peripheral venous blood and those in corpus cavernosal blood were similar (Koehn et al., 1993). These data suggested that VIP per se does not cause erection but could be acting in synergy with other vasodilating agents. On the other hand, coadministration of VIP and SIN-1 to human isolated cavernous arteries and cavernous smooth muscle did not cause any additive effect (Hempelmann et al., 1995). Numerous nitric oxide synthase and VIP-containing axons have, however, been identified in human penis. More than 50% of the perivascular nerve fibers and more than 90% of the trabecular nerve fibers within the corpus cavernosum stain positively for both NOS and VIP, whereas NOS/VIP-immunoreactivity is reduced in diabetes or absent in case of lesion of the cavernous nerve, supporting the concept that NO and VIP act as neural co-mediators of penile erection in humans (Ehmke et al., 1995). In a similar study, the distribution of cholinergic nerves in the human corpus cavernosum and spongiosum has been characterized using antibodies to the vesicular acetylcholine transporters, with this distribution compared to the distribution of hemoxygenases-1 and -2, neuronal and endothelial NO synthases, VIP, and vasoactive tyrosine. Vesicular acetylcholine transporters, NOS, and VIP were found to be localized in the same nerve terminals within the human corpus cavernosum and spongiosum (Hedlund et al., 2000). The role of VIP in penile tumescence and erection and rigidity was studied recently in a clinical study on adult males. Plasma levels of VIP were measured in the systemic and cavernous blood of 54 healthy adult male volunteers who were exposed to visual and tactile erotic stimuli in order to elicit penile tumescence and erection. In order to evaluate the role of VIP in detumescence, 16 volunteers were permitted to masturbate and ejaculate, and blood was then again withdrawn from the cavernous meshwork and the cubital vein. VIP levels were registered within the normal physiological range from 3.0 to 30 pmol/l and there was no increase in VIP plasma levels in the systemic and cavernous blood when the flaccid penis became rigid. Conversely, during penile detumescence, mean cavernous VIP level increased to 11.9 T 7.8 pmol/ l (baseline: 8.6 T 3.0 pmol/l), whereas VIP remained unaltered in the systemic circulation. Following ejaculation, mean VIP level

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in the cavernous blood was elevated to 25.3 T 10.9 pmol/l, whereas in the systemic blood no significant changes were registered, supporting the hypothesis that VIP plays a functional role in the mechanism of male sexual arousal (Becker et al., 2002). Thus, the exact role of VIP in the penis has not, as yet, clearly been established. If further studies will clarify its role, VIP receptors could represent a therapeutic target in male sexual dysfunction. 5. Drugs and therapy 5.1. Phosphodiesterase-5 as therapeutic target Phosphodiesterases (PDEs) catalyze the hydrolysis of the second messengers cAMP and cGMP that are responsible for activating a pathway of signaling events leading to smooth muscle relaxation. Both these second messengers are involved in signal pathways within the corpus cavernosum. The protein superfamily of cyclic nucleotide PDEs can be subdivided into at least 11 families of structurally and functionally related enzymes. So far, several isoforms have been characterized, all differing in their primary structures, specificity for cAMP and cGMP, mechanisms of regulation, and tissue distributions (Beavo, 1995; Polson & Strada, 1996; Dousa, 1999; Kuthe et al., 1999, 2000, 2001; Fawcett et al., 2000; Hetman et al., 2000; Soderling & Beavo, 2000). In human cavernous tissue, PDE3, a cAMP-dependent phosphodiesterase that is inhibited by cGMP; PDE4, a cAMP-specific phosphodiesterase; and PDE5, a cGMP-specific phosphodiesterase have been identified (Ballard et al., 1998; Bivalacqua et al., 1999; Kuthe et al., 2000, 2001). Molecular studies have demonstrated the presence of several other isoforms but functional studies have shown a role for PDE3 and PDE5 only (Ballard et al., 1998; Stief et al., 1998; Kuthe et al., 2000, 2001). Three different isoforms of PDE5 have been cloned in human penile tissue (Lin et al., 2000). Two isoforms were identical to PDE5A1 and PDE5A2, previously isolated from non-penile tissues, while the third isoform PDE5A3 was new. Expression of this isoform was present in tissues with a smooth muscle or cardiac muscle component. Recently, the 3-dimensional structures of the catalytic domain (residues 537 –860) of human PDE5 complexed with the 3 drug molecules sildenafil (Viagra), tadalafil (Cialis), and vardenafil (Levitra) have been determined (Sung et al., 2003) and these structures will provide opportunities to design further potent and selective PDE inhibitors with improved pharmacological profiles. The purpose of this section was to briefly introduce the oral PDE5 inhibitor sections that follow, in order to give a little insight into the complex field of phosphodiesterases. There are several reviews on the phosphodiesterase family that the reader can consult (Gupta et al., 2005; Jeon et al., 2005; Rybalkin et al., 2003). 5.1.1. Sildenafil Sildenafil is a highly selective inhibitor of PDE type 5 (Boolel et al., 1996; Godenberg, 1998). It enhances NOmediated relaxation of human corpus cavernosum in vitro

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(Ballard et al., 1998; Stief et al., 1998). By inhibiting phosphodiesterase, sildenafil increases the intracellular concentration of cyclic GMP, causing amplification of the endogenous NO-cGMP signaling pathway. Sildenafil was the first drug to be marketed for oral treatment of ED. As a result, several studies on its efficacy, safety, and tolerability have been published. In 2001, a systematic review and meta-analysis of 21 trials was performed. The meta-analysis showed that in all studies there was a statistically significant improvement in erectile or sexual function in patients using sildenafil compared with a placebo, demonstrating that sildenafil is an effective treatment for ED. However, it also suggested that the number needed to treat may be higher because many trial participants had some baseline erectile function (Burls et al., 2001). In 2002, there were a further 2 clinical updates on sildenafil. In 1 study, the data considered were pooled from 11 double-blind, placebo-controlled, flexible-dose (taken as needed) studies performed to assess efficacy (N = 2667) of sildenafil in men (aged 23– 89 years) with ED of broad-spectrum etiology who were not receiving concomitant nitrate therapies. A significantly improved erectile function was demonstrated for sildenafil compared with placebo for all efficacy parameters analyzed, regardless of patient age, race, body mass index, ED etiology, ED severity, ED duration, or the presence of various co-morbidities. Long-term effectiveness was assessed in 3 open-label extension studies. Approximately 95% of patients that continued long-term therapy (1– 3 years) with sildenafil reported that they were satisfied with the effect of treatment on their erections, and that treatment had improved their ability to engage in sexual activity (Carson et al., 2002). The only absolute contraindication from sildenafil is the concurrent use of nitrates (Padma-Nathan et al., 2002). A further retrospective analysis evaluated a sample of 152 depressed men with ED who were enrolled in a clinical trial of sildenafil. In this study, it was shown that erectile function changes were associated with improved mood and quality of sexual life, which resulted in improved partner satisfaction, family life, and overall life satisfaction (Rosen et al., 2004). Another issue addressed in clinical studies on sildenafil is whether the use of sildenafil can improve sexual function in healthy people. In a specific doubleblind, placebo-controlled trial clinical trial on 60 young healthy men, it was shown that sildenafil does not improve sexual function. Conversely, in this limited single-dose home study, sildenafil appeared to reduce the post-orgasmic refractory timelapse, suggesting that sildenafil could be used in premature ejaculation. However, at the present there are no controlled studies to support this hypothesis (Mondaini et al., 2003). Side effects associated with sildenafil use include headache, flushing, dyspepsia, and rhinitis and abnormal vision due to the inhibitory effect of sildenafil on PDE6. Long-term efficacy and safety of sildenafil have been also assessed in a large number of patients. In an open-label (with 36- or 52-week extension) study involving 1008 ED patients receiving doses in the range 25– 100 mg, it was demonstrated that about 90% of patients treated with sildenafil had improved their erections. The most commonly reported adverse events were headache, flushing, dyspepsia, and rhinitis, whereas reports of blurred vision were

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similar to that reported in previous clinical trials, indicating that long-term therapy does not diminish the efficacy of sildenafil in ED patients and remains well tolerated (Steers et al., 2001). The efficacy and tolerability of sildenafil was also demonstrated to be well maintained in older patients. In a specific analysis, it was shown that nearly two thirds of older subjects had a good response to the drug and no adverse effect was specific to older patients. However, a high prevalence of diabetes is one of the causes of lower efficacy rate of sildenafil in older men (Tsujimura et al., 2002). A possible efficacy of sildenafil has been tested also in females. This randomized, double-blind cross-over, placebocontrolled study was conducted at the Family Planning Centre of the Group for Sexological Research of the University of Catania, Italy. The study was performed on 68 healthy volunteer women aged 19– 38 years who were asymptomatic for sexual disorders. The study consisted of a 4-week sildenafil treatment period, 2 weeks of washout, and a 4-week placebo treatment, in 2 possible sequences: sildenafil 50 mg, washout, placebo; or placebo, washout, sildenafil 50 mg. Sildenafil significantly improved arousal, orgasm, and enjoyment when compared to the placebo effect. The adverse events were transient and mild or moderate. These data suggest that sildenafil acts on different sexual pathways in healthy women, improving their sexual experience. However, additional studies are required to define the use of sildenafil in a clinical setting (Caruso et al., 2003). While the only clear contraindication for sildenafil is the concomitant use of nitrates, one of the major concerns to the use of sildenafil and of any other PDE5 inhibitor is the possible increased risk of myocardial infarction and cardiovascular death. A lack of association of sildenafil use with increased risk of MI or cardiovascular death was shown in a meta-analysis study. The study took into account pooled data regarding the incidence of myocardial infarction and cardiovascular death from more than 120 clinical trials of sildenafil citrate conducted from 1993 to 2001. This analysis showed that the rates of MI and cardiovascular death were low and comparable between men treated with sildenafil and those treated with placebo (Mittleman & Glasser, 2003). Sildenafil citrate does not affect macroscopic and microscopic seminal parameters in addition to the acrosome reaction. Sperm zone pellucid binding results were increased by sildenafil treatment and it has been suggested that Viagra could be used to enhance sperm motility and binding to the oocyte, especially during fertility treatments (Du Plessis et al., 2004). Sildenafil has been also shown to have an effect on the human retina. Indeed, in a randomized, double-blind, placebo-controlled, 2-way crossover clinical study in 12 healthy male volunteers, following a single administration of 100 mg of sildenafil, there was a significant increase in retinal venous diameters and retinal blood flow when compared to placebo. Sildenafil at the dose used did not affect intraocular pressure or flicker-induced retinal vasodilatation (Polak et al., 2003). Sildenafil could also precipitate migraine attacks in ED patients suffering from migraine. Indeed, in 12 patients with migraine without aura, a doubleblind, placebo-controlled crossover study was performed, in which placebo or 100 mg sildenafil was administered orally on

2 separate days. The study involved measurement of blood flow velocity in the middle cerebral artery and regional cerebral blood flow in the territory of the middle cerebral artery was measured using single photon emission computed tomography and xenon 133 inhalation. Headache response, tenderness of pericranial muscles, blood pressure, and heart rate were also measured repeatedly. Migraine attack was induced by sildenafil in 10 of 12 migraine patients and by placebo in 2 of 12 patients (Kruuse et al., 2003). In prescribing and using sildenafil, it should be also taken into account that erythromycin modifies the pharmacokinetics of sildenafil by inhibiting the cytochrome P450 isozyme 3A4 (CYP3A4)-mediated first-pass metabolism, even though in the same study azithromycin did not affect the pharmacokinetics of sildenafil. Thus, it is necessary that caution should be taken in administering sildenafil with drugs known to interfere with CYP3A4 because sildenafil is predominantly metabolized by the CYP3A4 isozyme (Muirhead et al., 2002a, 2002b). Sildenafil’s pharmacokinetic profile is also affected by age and by renal and hepatic impairment, suggesting that a lower starting dose of 25 mg should be considered for patients with severely compromised renal or hepatic function (Muirhead et al., 2002a, 2002b). 5.1.2. Tadalafil Tadalafil is a potent inhibitor of PDE5, has a half-life of 17.5 h, and a long lasting effect, up to 36 h, after dosing. Tadalafil is effective and well tolerated. In a recent update, the outcomes of clinical trials conducted worldwide in 174 centers from April 1999 to February 2003 were analyzed. All the studies considered were randomized, double blind, and placebo controlled. The study considered a total of 2102 patients, made up of 638 placebo, 321 tadalafil 10 mg, and 1143 tadalafil 20 mg. To be included in these studies, patients were required to make at least 4 attempts at sexual intercourse during a 4-week treatment-free initial period. Patients took the treatment as needed before sexual intercourse with no restrictions on food intake or the timing of sexual activity. The mean age was 56 (22 – 88) years with 88% of the patients having ED lasting at least 1 year. The most common comorbities were hypertension (29%), diabetes mellitus (20%), and hyperlipidemia (16%). Patients treated with tadalafil showed a highly significant effect over placebo at both doses used (10 or 20 mg) on the International Index of Erectile Function, the Sexual Encounter Profile (SEP-2, SEP-3) score, and the global assessment question (Carson et al., 2004). In another retrospective analysis of pooled data from 12 placebo-controlled trials, the efficacy and safety of tadalafil for the treatment of ED in men with diabetes, compared with ED men without diabetes, were evaluated. The results of this analysis showed that there is a more severe baseline ED in men with diabetes and that tadalafil was efficacious and well tolerated. However, compared to other phosphodiesterase 5 inhibitors, the response to tadalafil was lower in men with diabetes than in men without diabetes (Fonseca et al., 2004). In another specific clinical trial, efficacy in patients with ED following bilateral nerve sparing radical retropubic prostatectomy was assessed. In this study, 12 weeks of treatment with tadalafil 20 mg on demand showed a good

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efficacy, causing a significant increase in IIEF, SEP2, and SEP3, as well as of other parameters (Montorsi et al., 2004a). One peculiar side effect that has been recently reported is a transient global amnesia after the first use of tadalafil, suggesting that PDE5 inhibitors can exert adverse effects on the central nervous system and thus it is important that the physician take this into account if the patient has any neurological disturbance (Schiefer & Sparing, 2005). As for sildenafil, other possible therapeutic uses of tadalafil have been explored. In a recent study, it has been shown that 20 mg tadalfil, given on alternate days in patients with an increased cardiovascular risk, causes improved endothelial function regardless of their degree of ED. The study showed a significant effect of tadalafil versus placebo on brachial artery flow-mediated dilation. However, this was a small study (32 patients); thus, no definitive conclusion can be drawn (Rosano et al., 2005). Through a study performed in human volunteers, it was shown that therapeutic concentrations of tadalafil do not produce clinically significant changes in the clearance of drugs metabolized by CYP3A. Indeed, in a recent clinical study, the pharmacokinetics of midazolam and lovastatin, 2 different CYP3A substrates, was found to be virtually unchanged after tadalafil co-administration (Ring et al., 2005). Recently, as for vardenafil and sildenafil, the possible interaction of tadalafil with a-antagonists was studied in specific clinical trial. Indeed, a-antagonists are extremely important in the treatment of benign prostate hypertrophy. Tadalafil (20 mg) augmented the hypotensive effect of doxazosin by producing a mean maximal decrease in standing systolic BP that was significantly greater than placebo. Conversely, in subjects treated with tamsulosin, 10 and 20 mg of tadalafil produced mean maximal systolic blood pressure reductions that were similar to placebo, suggesting that caution should be used when patients are using doxazosin (Kloner et al., 2004). Finally, it should be taken into account that commercial grapefruit juice, given as a single normal amount (e.g., 200 – 300 mL), causes irreversible inactivation of intestinal cytochrome P450 3A4. This means it can interact with sildenafil, tadalafil, or vardenafil, causing serious systemic vasodilatation, especially when combined with a nitrate therapy (Bailey & Dresser, 2004). 5.1.3. Vardenafil Vardenafil is a potent, selective inhibitor of PDE5. Its structure is similar to that of sildenafil. In vitro, vardenafil selectively inhibits human PDE5 purified from human corpus cavernosum and displays a significantly reduced IC50 when compared to sildenafil and tadalafil. However, this increased potency in vitro does not translate to a greater therapeutic effect over tadalfil and sildenafil in vivo. The only advantage that vardenafil has over sildenafil is that it does not inhibit phosphodiesterase-6 and thus alter color perception, a rare side effect that sometimes occurs with sildenafil. Vardenafil is well absorbed after a single dose administration, with maximum plasma concentration reached within 0.6– 0.9 h. However, an effect of vardenafil has been shown to be present even earlier than 0.6 h in specific clinical trials (Padma-Nathan

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et al., 2003; Montorsi et al., 2004b). Vardenafil absorption is not modified by a meal containing 30% of calories as fat when compared to a fasting state, whereas a high-fat meal reduced the rate of absorption, increasing the time to maximum plasma concentration to 1 hr and reducing the C max to 18% (Rajagopalan et al., 2003). Vardenafil is metabolized by cytochrome P450 3A4, and thus inhibitors of this enzyme can increase plasma levels of vardenafil. As for sildenafil, concomitant use of vardenafil with nitrates is contraindicated, whereas concomitant treatment with a-antagonists should be only initiated when the patient has been stabilized on aantagonist therapy. Vardenafil was tested in clinical trials prior to and after its launch on the market. These were mostly randomized, double-blind studies with fixed or flexible dose design. In all these studies, vardenafil was demonstrated to have significant activity over placebo and most patients receiving vardenafil indicated that their erections were returned to almost normal erectile function (Hellstrom et al., 2002). Vardenafil has been also tested in a specific, double-blind, and controlled fixed-dose study involving 452 diabetic patients. Indeed, diabetic patients generally display a reduced response to PDE5 inhibitors. This study showed that after 12 weeks of therapy, different indices of erectile function (IIEF, SEP-2, SEP3, and GAQ) were significantly improved compared to placebo. The effect was shown to be independent of baseline ED severity, glycemic control, and unrelated to the type of diabetes. Vardenafil efficacy has also been tested in patients that underwent radical prostatectomy and depressive symptomatology. In these specific trials, it was shown that vardenafil (20 mg for 12 weeks) can achieve 70% success in restoring the erectile function in patients that have undergone nerve sparing radical retropubic prostatectomy. Similarly, in men with mild major depressive disorders that were not receiving psychotherapy or antidepressant drugs, treatment with vardenafil for 12 weeks improved their erectile function. Overall, vardenafil has been administered to more than 4000 people and the most common adverse events are headache, flushing, and rhinitis, with incidences between 10% and 16%. Concerning the cardiovascular effects that always represent a major concern for the use of these drugs, a pooled analysis of data from 7 clinical trials showed that in patients receiving concomitant antihypertensive therapy, vardenafil treatment causes a small reduction in systolic and diastolic blood pressure, ranging between 3 and 4 mmHg, and no substantial change in heart rate. All the other cardiovascular events monitored (angina, syncope, ECG alterations, and hypotension) were unrelated to vardenafil dose. The safety of vardenafil has been also tested on ischemic events in man with stable coronary disease. The drug did not exacerbate the ischemic response at a dose of 10 or 20 mg (Thadani et al., 2002, 2003), nor did it modify QT interval (Morganroth et al., 2004). 5.2. Intracavernous therapy 5.2.1. Prostaglandin E1 Alprostadil is a synthetic prostaglandin E1 (PGE, alprostadil) with myorelaxant activity, traditionally used for the

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management of peripheral arterial occlusive disease and for the maintenance of patency of ductus arteriosus Botalli in the socalled ‘‘bleu babies.’’ The relaxant effect of PGE1 on human corpus cavernosum in vitro was initially described in 1979. In further studies, PGE1 was shown to elicit a dose-dependent relaxation in corpus cavernosum strips pre-contracted with noradrenaline (Hedlund & Andersson, 1985). A few months later, the first reports on self-injection of PGE1 as a treatment for ED were presented (Adaikan et al., 1986a, 1986b). Later on, alprostadil became increasingly popular and in the mid1990s was regarded as an established diagnostic and therapeutic tool for ED (Linet et al., 1996; Von Eyden et al., 1993). Typical dosing of alprostadil is 1 –60 Ag, although most authors consider 20 Ag as the maximum dosage. As a matter of fact, above 20 Ag, a plateau of erectile response is reached and only a non-linear dose-dependent effect can be achieved. At this dosage, alprostadil is effective in 73% of cases of ED across all etiologies and is superior to older injectable drugs such as papaverine or papaverine –phentolamine, being effective in 53% and 68% of cases, respectively, whereas the minor risk of priapism is 0.25% for PGE1 versus 6.8% and 6% for papaverine and papaverine – phentolamine, respectively (Porst, 1996). With long term use, the most frequently reported side effects are nodules of tunica albuginea at the site of injection and fibrosis of corpus cavernosum (0.8% with PGE1 vs. 5.7% and 12.4% with papaverine and papaverine –phentolamine, respectively). Despite the good effectiveness and safety profile, high dropout rates, up to 40%, have been reported in most series (Porst, 1996). Studies aimed to clarify the reasons for such a low rate of patient satisfaction showed that objective side effects were not the main factor. More importantly, low motivation and poor satisfaction with the quality of the injection-induced erection (mainly for the bothersomeness of the administration method) can make the patient unable to fully accept the treatment, hence it is eventually ineffective in improving self-esteem (Lehmann et al., 1999). Alprostadil maintained its role of market prescriptions leader until 1998, when sildenafil was introduced in the clinical practice. Currently, in the ‘‘oral era,’’ injectable PGE1 is a second-line therapeutic option only proposed when oral drugs are contraindicated (e.g., patients taking nitrates) or are producing ineffective results. Additionally, PGE1 remains the election drug in the dynamic penile color Doppler diagnostic test to evaluate vascular function of corpora cavernosa. It is worth noting that a transurethral formulation of alprostadil has been on the market since 1997, and that recently a topical formulation has been produced. The intraurethral formulation has proven to be less effective than the intracavernous formulation, with a high rate of local side effects such as penile pain and urethral bleeding. This unfavorable profile produced a very high dropout rate of patients (about 75%). At present, intraurethral alprostadil has a very limited role in clinical practice. Topical alprostadil has proven in phase II studies to be promising in terms of efficacy, compliance, and tolerability, with minimal local erythema being the most frequent side effect. Further studies are warranted to fully evaluate this novel therapeutic option.

5.2.2. Other therapies Other injectable agents include papaverine, phentolamine, Vasoactive Intestinal Polypeptide (VIP), atropine sulfate, and a few others. Currently, all of them are obsolete or considered too experimental to be used as monotherapy. However, a combination of 2, 3, or 4 injectable drugs (bi- and tri-mix) is still a valuable option in patients who are non-responders to oral drugs or to the maximum dosage of alprostadil (Baniel et al., 2000; Steers, 2003). The most widely diffused cocktail is a tri-mix of alprostadil, papaverine, and phentolamine, reputed to be the most powerful drug treatment for ED available so far and reserved for severe cases of non-responsiveness to other treatments, for example, patients with ED post-radical prostatectomy (Baniel et al., 2001). 6. Conclusions and future direction ED is a complex multifactorial event that is linked to many morbidity factors, of which the most important is age. The introduction on the market in 1998 of sildenafil as an oral remedy for ED has opened an entirely new field of research that has been flourishing in the past 5 years. Indeed, information on the pathophysiology of erection has increased enormously in the past 5 years. As has been described and discussed in the above sections, we now have much information and are in the process of identifying new therapeutic targets that should allow us to cure those cohorts of patients that are non-responsive to PDE5 therapy. From this review, the reader has most likely realized that experimental observations made in rat, mouse, or rabbit models turn out to be not always true in man, hence particular caution must be used before extrapolating animal data to humans concerning the possible role played by different mediators. Furthermore, it appears clear that clinical data mostly rely on efficacy as the primary endpoint on validated questionnaires since at present there are no objective parameters that can be measured which accurately represent the relative potency of the drug under examination. Thus, it is required that basic and clinical research identifies and validates new endpoints that may allow a better understanding of the biological activity of a drug on the market, as well as the validation of new therapeutic targets. Finally, it must be said that lower urinary tract symptoms (LUTS), which are extremely common in men, have been suggested to be related to ED in the aging male (Braun et al., 2003). A close correlation between ED and LUTS was found in a recent study performed on 1274 European men with lower urinary tract symptoms. ED strongly correlated with LUTS severity, with 55% of people with mild symptoms reporting ED and 70% of people with severe symptoms reporting ED. Reduced ejaculation was also significantly related to LUTS severity, with an incidence of 55% in mild LUTS and 68% in severe LUTS. Interestingly, this study also showed that pain/discomfort on ejaculation was only related to lower urinary tract symptom severity, being associated with 31% of patients with mild LUTS, as opposed to 7% in severe LUTS. Thus, these data strongly imply that ED and reduced ejaculation are highly

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prevalent in men with LUTS (Vallancien et al., 2003). In a more recent survey conducted to investigate the relationship between LUTS and sexual problems in both men and women, a detailed questionnaire was mailed to a random sample of 15,000 men and women aged 40 – 65 years. LUTS and sexual function were assessed by validated symptom scales. Of the 15000 questionnaires, 7741 were considered valuable and included in the analysis. The logistic regression analysis of factors relating to erection problems and satisfaction with sex life in men and sexual function in women indicated that LUTS are an independent risk factor for sexual dysfunction in both men and women aged 40 – 65 years. All these results highlight the clinical importance of not only evaluating LUTS in patients with sexual dysfunction (Hansen, 2004), but also of considering new etiopathologic models of ED that can explain, in terms of molecular cascade, the epidemiological association of LUTS and ED. This perspective is expected to affect new hypotheses and future research direction and opens an intriguing scenario where a single therapeutic intervention may treat or even prevent 2 prevalent, age-related, quality-of-life-reducing diseases like LUTS and ED. Connecting both diseases to a common pathway would probably give us the ‘‘reading-key’’ of 2 of the most challenging conditions of modern medical sciences and would refresh the often forgotten postulate that a man must be considered as a whole, and not as a ‘‘multi-organ’’ machine only. In conclusion, large gaps in research on ED has been filled in these past 7 years and our knowledge on the pathophysiology of ED and on male sexual problems in general have expanded enormously. However, there are still many unanswered questions that need to be addressed and more efforts need to be made in order to improve drug design and therapy. In addition, even though ED is not a life-threatening disease, it is an important factor to be considered in the global assessment of quality of life and may represent an early sign of cardiovascular disease. References Adaikan, P. G., Kottegona, S. R., Ratnam, S. S. (1986). A possible role for prostaglandin E1 for organic impotence. Abstract Book Second World Meeting on Impotence. Prague, Czechoslovakia, February 6, 1986. Adaikan, P. G., Kottegoda, S. R., Ratnam, S. S. (1986). Is vasoactive intestinal polypeptide the principal transmitter involved in human penile erection? J Urol 135, 638 – 640. Andersson, K.-E. (2001). Pharmacology of penile erection. Pharm Rev 53, 417 – 450. Ando, S., Rubens, R., Rottiers, R. (1984). Androgen plasma levels in male diabetics. J Endocrinol Invest 7, 21 – 24. Angulo, J., Cuevas, P., La Fuente, J. M., Pomerol, J. M., Ruiz-Castane, E., Puigvert, A., et al. (2002). Regulation of human penile smooth muscle tone by prostanoid receptors. Br J Pharmacol 136, 23 – 30. Bailey, D. G., Dresser, G. K. (2004). Interactions between grapefruit juice and cardiovascular drugs. Am J Cardiovasc Drugs 4, 281 – 297. Ballard, S. A., Gingell, C. J., Tang, K., Turner, L. A., Price, M. E., Naylor, A. M. (1998). Effects of sildenafil on the relaxation of human corpus cavernosum tissue in vitro and on the activities of cyclic nucleotide phosphodiesterase isozymes. J Urol 159, 2164 – 2171. Bancroft, J., Munoz, M., Beard, M., Shapiro, C. (1995). The effects of a new alpha-2 adrenoceptor antagonists on sleep and nocturnal penile tumescence

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