Σκοπός: Να διερευνηθεί η επίδραση της καθημερινής χορήγησης σιλδεναφίλης στα επίπεδα διαλυτών δεικτών της ενδοθηλιακής λειτουργίας σε άνδρες με στυτική δυσλειτουργία. Μέθοδοι: Ασθενείς πάνω από 18 ετών με στυτική δυσλειτουργία αγγειακής αιτιολογίας για πάνω από 6 μήνες, είτε μόνη είτε σε συνδυασμό με νοσολογικές καταστάσεις ισχυρά συσχετιζόμενες με ενδοθηλιακή δυσλειτουργία όπως σακχαρώδης διαβήτης/μεταβολικό σύνδρομο, υπέρταση και στεφανιαία νόσος, έλαβαν σιλδεναφίλη 25 mg ημερησίως από του στόματος για 4 εβδομάδες. Δείκτες της ενδοθηλιακής λειτουργίας μετρήθηκαν στο πλάσμα στην αρχή και το τέλος της θεραπείας χρησιμοποιώντας τυπικές μεθόδους και εμπορικά διαθέσιμα υλικά. Αποτελέσματα: 112 άνδρες με μέση ηλικία (SD) 60,6 (7,3) έτη ολοκλήρωσαν το θεραπευτικό πρωτόκολλο. Η χορήγηση 25 mg σιλδεναφίλης καθημερινά για 4 εβδομάδες μείωσε σημαντικά τα επίπεδα της ενδοθηλίνης-1 σε σύγκριση με την αρχή της θεραπείας (2,83 ± 1,63 έναντι 3,24 ± 1,90 pg/ml, p<0,001). Σημαντικές αλλαγές παρατηρήθηκαν επίσης για το οξείδιο του αζώτου (ΝΟ) (35,12 ± 21,14 έναντι 31,91 ± 16,28 pmol/lt, p=0,01), τα επίπεδα της cGMP (3,79 ± 2,37 έναντι 2,70 ± 1.34 pmol/ml, p<0,001) και τον παράγοντα von Willebrand (956,08 ± 514,25 έναντι 1007,42 ± 466,25 mU/ml) αλλά όχι και για τους άλλους δείκτες που μετρήθηκαν (θρομβομοδουλίνη και Ε-σελεκτίνη). Η στυτική λειτουργία βελτιώθηκε επίσης. Συμπεράσματα: Η σιλδεναφίλη σε καθημερινή χορήγηση βελτιώνει την ενδοθηλιακή λειτουργία όπως αυτή εκτιμάται με τα επίπεδα βιολογικών δεικτών σε ασθενείς με στυτική δυσλειτουργία. Αυτά τα αποτελέσματα συμφωνούν με άλλες μελέτες που δείχνουν όμοια αποτελέσματα με θεραπεία με αναστολείς της φωσφοδιεστεράσης 5. Η κλινική σημασία των αποτελεσμάτων αυτών χρήζει περαιτέρω διερεύνησης. / Objective: To investigate the impact of daily sildenafil on levels of soluble molecular markers of endothelial function in men with erectile dysfunction. Methods: Patients over 18 years of age with erectile dysfunction of vascular aetiology for more than 6 months, either alone or in combination with disease states strongly associated with endothelial dysfunction such as diabetes/metabolic syndrome, hypertension and coronary artery disease, received sildenafil 25 mg orally for 4 weeks. Markers of endothelial function were measured in plasma at baseline and end-of-treatment using standard methods and commercially available kits. Results: 112 men with mean (SD) age of 60.6 (7.3) years completed the protocol. Sildenafil 25mg daily for 4 weeks significantly reduced endothelin-1 levels compared to baseline (2.83 ± 1.63 vs. 3.24 ± 1.90 pg/ml, p<0.001). Significant changes were also observed for nitric oxide (35.12 ± 21.14 vs. 31.91 ± 16.28 pmol/lt, p=0.01) and cyclic guanosine monophosphate (3.79 ± 2.37 vs. 2.70 ± 1.34 pmol/ml, p<0.001) and von Willebrand factor (956.08 ± 514.25 vs. 1007.42 ± 466.25 mU/ml) levels but not for the other biomarkers measured (thrombomodulin and E-selectin). Erectile function was significantly improved. Conclusions: Daily sildenafil improves endothelial function as assessed by levels of biomarkers of endothelial function in patients with erectile dysfunction. This is in agreement with other studies showing similar benefits with phosphodiesterase 5 inhibitor treatment. The clinical implications of this finding need further investigation.

A disfunção erétil (DE) tem alta prevalência entre hipertensos e tem sido considerada marcador precoce de risco cardiovascular. A presença e gravidade da DE bem como a resposta clínica aos inibidores da fosfodiesterase tipo 5 (PDE5) parecem depender da biodisponibilidade do óxido nítrico (NO) endotelial e da extensão da doença aterosclerótica. O objetivo deste estudo foi avaliar a resposta clínica da vardenafila usada em dois regimes terapêuticos em hipertensos com DE vasculogênica e sem doença cardiovascular maior, correlacionando a gravidade da DE e a eficácia da vardenafila com dados antropométricos, laboratoriais, escore de risco cardiovascular e parâmetros vasculares funcionais e estruturais. A resposta clínica à vardenafila nos dois regimes foi avaliada conforme o percentual de respostas positivas à questão 3 do Perfil do Encontro Sexual (PES3). Os parâmetros vasculares considerados foram a espessura médio-intimal (EMI) da carótida comum, a dilatação mediada pelo fluxo (DMF) da artéria braquial e a dilatação nitrato-mediada (DNM). Foram incluídos 100 homens hipertensos com idade entre 50 e 70 anos, sendo 74 portadores de DE vasculogênica e 26 com função erétil normal que serviram de grupo controle. Nos pacientes com DE, o índice de massa corporal, relação cintura-quadril, EMI da carótida, níveis séricos de triglicerídeos, colesterol total e LDL foram significativamente maiores que no grupo controle. Após o uso de vardenafila on demand (fase 1), os pacientes com mais de 50% de respostas positivas ao PES3 ou 50% de respostas afirmativas e um incremento de 6 pontos ou mais em relação ao Índice Internacional de Função Erétil (IIEF-FE) basal e/ou resposta positiva a Questão de Avaliação Global (QAG), foram considerados respondedores. O escore do IIEF-FE basal se correlacionou negativamente com a EMI da carótida (r=-0,48, P<0,001) e com o escore de Framingham (r= -0,41, P<0,001) no grupo com DE. Houve forte correlação positiva entre a resposta clínica à vardenafila com a DMF (r= 0,70, P<0,001), que não se observou entre o sub-grupo de diabéticos. Os 35 pacientes considerados não-respondedores na fase 1 foram randomizados e, em desenho duplo-cego, receberam vardenafila ou placebo diariamente durante cinco semanas, podendo usar 10 mg de vardenafila uma hora antes da atividade sexual (fase2). Houve resposta clínica positiva em 38,8% dos que receberam a vardenafila na fase 2 e esta resposta se correlacionou com a frequência sexual (r= 0,68, P<0,01) e com o escore de Framingham (r= -0,65, P<0,01), com a EMI da carótida (r= -0,61, P=0,01) e com o LDL-colesterol (r= -0,64, P<0,01). A vardenafila foi bem tolerada em ambos os regimes terapêuticos. Concluímos que nessa amostra de hipertensos, a gravidade da DE foi relacionada a parâmetros vasculares estruturais (EMI), enquanto a resposta clínica à vardenafila on demand foi mais diretamente dependente da função vascular momentânea (DMF). Houve benefício na utilização de vardenafila diariamente com o objetivo de resgatar a eficácia do inibidor quanto à melhora do desempenho sexual. A falta de eficácia clínica ao inibidor da PDE5 em ambos os regimes terapêuticos pode servir como marcador clínico que identifica homens hipertensos com um risco cardiovascular aumentado. / Erectile dysfunction (ED) is a high prevalent disease in hypertensive subjects and has been considered an early cardiovascular risk marker. EDs presence and severity, as well as clinical response to phosfodiesterase type 5 (PDE5) inhibitors, vary according to nitric oxide (NO) availability and atherosclerosis extension. We investigated whether vasculogenic ED severity and clinical response to vardenafil used on demand or continuously were associated with structural and functional vascular changes in patients with uncomplicated hypertension. Our main efficacy criterion was per patient percentage of positive answers on Sexual Encounter Profile question 3(SEP3). Vascular parameters considered were intima-media thickness (IMT), flow-mediated dilation (FMD) on brachial artery and nitrate-mediated dilation. A total of 100 hypertensive men aging between 50 and 70 years were included. Among these patients, 74 had vasculogenic ED and 26 presented normal erectile function according to erectile domain of International Index of Erectile Function (IIEF-EF). Among those with ED, body mass index, waist-rip ratio, carotid IMT, triglycerides, total cholesterol and LDL-cholesterol were significantly higher than controls. After vardenafil on demand usage during phase 1, patients with more than 50% of positive answers on SEP3 or 50% and more than 6 points on IIEF basal score or positive answer to global evaliation question were considered responders. IIEF basal score correlated inversely with carotid IMT (r=-0.48, P<0.001) and with Framingham risk score (r= -0.41, P<0.001) in ED group. Clinical response to vardenafil strongly correlated with FMD (r= 0.70, P<0.001), except among diabetics. Non responders (n=35) on phase 1 were included on phase 2 when, after randomization, they received vardenafil 10 mg nightly or placebo during five weeks. Open vardenafil on demand were allowed one hour before sexual intercourse, and 38.8% of active group improved and became responders to vardenafil. Clinical response on phase 2 correlated with sexual frequency (r= 0.68, P<0.01), Framingham score (r= -0.65, P<0.01), carotid IMT (r= -0.61, P=0.01) and LDL-cholesterol (r= -0.64, P<0.01). We concluded that in hypertensive males with vasculogenic ED and no other clinical evidence of atherosclerosis, ED severity correlated with structural parameters (carotid IMT), while phosphodiesterase-5 effectiveness correlated with functional vascular aspects (brachial FMD). There were positive impact with continuous vardenafil on non responders to on demand regime and that could be an option to salvage strategy. Lack of PDE5 inhibitor efficacy in both therapeutic strategies could point out to higher cardiovascular risk and could be considered a useful clinical marker.

A disfunção erétil (DE) tem alta prevalência entre hipertensos e tem sido considerada marcador precoce de risco cardiovascular. A presença e gravidade da DE bem como a resposta clínica aos inibidores da fosfodiesterase tipo 5 (PDE5) parecem depender da biodisponibilidade do óxido nítrico (NO) endotelial e da extensão da doença aterosclerótica. O objetivo deste estudo foi avaliar a resposta clínica da vardenafila usada em dois regimes terapêuticos em hipertensos com DE vasculogênica e sem doença cardiovascular maior, correlacionando a gravidade da DE e a eficácia da vardenafila com dados antropométricos, laboratoriais, escore de risco cardiovascular e parâmetros vasculares funcionais e estruturais. A resposta clínica à vardenafila nos dois regimes foi avaliada conforme o percentual de respostas positivas à questão 3 do Perfil do Encontro Sexual (PES3). Os parâmetros vasculares considerados foram a espessura médio-intimal (EMI) da carótida comum, a dilatação mediada pelo fluxo (DMF) da artéria braquial e a dilatação nitrato-mediada (DNM). Foram incluídos 100 homens hipertensos com idade entre 50 e 70 anos, sendo 74 portadores de DE vasculogênica e 26 com função erétil normal que serviram de grupo controle. Nos pacientes com DE, o índice de massa corporal, relação cintura-quadril, EMI da carótida, níveis séricos de triglicerídeos, colesterol total e LDL foram significativamente maiores que no grupo controle. Após o uso de vardenafila on demand (fase 1), os pacientes com mais de 50% de respostas positivas ao PES3 ou 50% de respostas afirmativas e um incremento de 6 pontos ou mais em relação ao Índice Internacional de Função Erétil (IIEF-FE) basal e/ou resposta positiva a Questão de Avaliação Global (QAG), foram considerados respondedores. O escore do IIEF-FE basal se correlacionou negativamente com a EMI da carótida (r=-0,48, P<0,001) e com o escore de Framingham (r= -0,41, P<0,001) no grupo com DE. Houve forte correlação positiva entre a resposta clínica à vardenafila com a DMF (r= 0,70, P<0,001), que não se observou entre o sub-grupo de diabéticos. Os 35 pacientes considerados não-respondedores na fase 1 foram randomizados e, em desenho duplo-cego, receberam vardenafila ou placebo diariamente durante cinco semanas, podendo usar 10 mg de vardenafila uma hora antes da atividade sexual (fase2). Houve resposta clínica positiva em 38,8% dos que receberam a vardenafila na fase 2 e esta resposta se correlacionou com a frequência sexual (r= 0,68, P<0,01) e com o escore de Framingham (r= -0,65, P<0,01), com a EMI da carótida (r= -0,61, P=0,01) e com o LDL-colesterol (r= -0,64, P<0,01). A vardenafila foi bem tolerada em ambos os regimes terapêuticos. Concluímos que nessa amostra de hipertensos, a gravidade da DE foi relacionada a parâmetros vasculares estruturais (EMI), enquanto a resposta clínica à vardenafila on demand foi mais diretamente dependente da função vascular momentânea (DMF). Houve benefício na utilização de vardenafila diariamente com o objetivo de resgatar a eficácia do inibidor quanto à melhora do desempenho sexual. A falta de eficácia clínica ao inibidor da PDE5 em ambos os regimes terapêuticos pode servir como marcador clínico que identifica homens hipertensos com um risco cardiovascular aumentado. / Erectile dysfunction (ED) is a high prevalent disease in hypertensive subjects and has been considered an early cardiovascular risk marker. EDs presence and severity, as well as clinical response to phosfodiesterase type 5 (PDE5) inhibitors, vary according to nitric oxide (NO) availability and atherosclerosis extension. We investigated whether vasculogenic ED severity and clinical response to vardenafil used on demand or continuously were associated with structural and functional vascular changes in patients with uncomplicated hypertension. Our main efficacy criterion was per patient percentage of positive answers on Sexual Encounter Profile question 3(SEP3). Vascular parameters considered were intima-media thickness (IMT), flow-mediated dilation (FMD) on brachial artery and nitrate-mediated dilation. A total of 100 hypertensive men aging between 50 and 70 years were included. Among these patients, 74 had vasculogenic ED and 26 presented normal erectile function according to erectile domain of International Index of Erectile Function (IIEF-EF). Among those with ED, body mass index, waist-rip ratio, carotid IMT, triglycerides, total cholesterol and LDL-cholesterol were significantly higher than controls. After vardenafil on demand usage during phase 1, patients with more than 50% of positive answers on SEP3 or 50% and more than 6 points on IIEF basal score or positive answer to global evaliation question were considered responders. IIEF basal score correlated inversely with carotid IMT (r=-0.48, P<0.001) and with Framingham risk score (r= -0.41, P<0.001) in ED group. Clinical response to vardenafil strongly correlated with FMD (r= 0.70, P<0.001), except among diabetics. Non responders (n=35) on phase 1 were included on phase 2 when, after randomization, they received vardenafil 10 mg nightly or placebo during five weeks. Open vardenafil on demand were allowed one hour before sexual intercourse, and 38.8% of active group improved and became responders to vardenafil. Clinical response on phase 2 correlated with sexual frequency (r= 0.68, P<0.01), Framingham score (r= -0.65, P<0.01), carotid IMT (r= -0.61, P=0.01) and LDL-cholesterol (r= -0.64, P<0.01). We concluded that in hypertensive males with vasculogenic ED and no other clinical evidence of atherosclerosis, ED severity correlated with structural parameters (carotid IMT), while phosphodiesterase-5 effectiveness correlated with functional vascular aspects (brachial FMD). There were positive impact with continuous vardenafil on non responders to on demand regime and that could be an option to salvage strategy. Lack of PDE5 inhibitor efficacy in both therapeutic strategies could point out to higher cardiovascular risk and could be considered a useful clinical marker.

Ce projet de maitrise implique le développement et l’optimisation de deux méthodes utilisant la chromatographie liquide à haute performance couplée à la spectrométrie de masse en tandem (HPLC-MS/MS). L'objectif du premier projet était de séparer le plus rapidement possible, simultanément, 71 médicaments traitant la dysfonction érectile (ED) et 11 ingrédients naturels parfois retrouvés avec ces médicaments dans les échantillons suspectés d’être adultérés ou contrefaits. L'objectif du deuxième projet était de développer une méthode de dépistage permettant l'analyse rapide simultanée de 24 cannabinoïdes synthétiques et naturels pour une grande variété d'échantillons tels que les mélanges à base de plantes, des bâtons d'encens, de sérums et de cannabis. Dans les deux projets, la séparation a été réalisée en moins de 10 min et cela en utilisant une colonne C18 à noyau solide 100 x 2,1 mm avec des particules de 2,6 µm de diamètre couplée à un système MS avec trappe ionique orbitale fonctionnant en électronébulisation positive. En raison du nombre élevé de composés dans les deux méthodes et de l’émergence de nouveaux analogues sur le marché qui pourraient être présents dans les échantillons futurs, une méthode de dépistage LC-MS/MS ciblée/non-ciblée a été développée. Pour les deux projets, les limites de détection étaient sous les ng/mL et la variation de la précision et de l’exactitude étaient inférieures de 10,5%. Le taux de recouvrement à partir des échantillons réels variait entre 92 à 111%. L’innovation des méthodes LC-MS/MS développées au cours des projets est que le spectre de masse obtenu en mode balayage lors de l'acquisition, fournit une masse exacte pour tous les composés détectés et permet l'identification des composés initialement non-ciblés, comme des nouveaux analogues. Cette innovation amène une dimension supplémentaire aux méthodes traditionnellement utilisées, en permettant une analyse à haute résolution sur la masse de composés non-ciblés. / This master’s project involved the development and optimization of two rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) methods. The objective of the first project was to simultaneously separate, as rapidly as possible, 71 erectile dysfunction (ED) treatment drugs and 11 natural ingredients sometimes found alongside ED drugs present in suspected adulterated or counterfeit samples. The objective of the second project was to develop a screening method allowing rapid, simultaneous analysis of 24 synthetic and natural cannabinoids for a wide variety of samples such as herbal smoking mixtures, incense sticks, serums and Cannabis plant material. In both projects, the separations were achieved in ≤ 10 min using 2.6 µm fused-core C18 particles packed into a 100 x 2.1 mm column coupled to an LTQ Orbitrap XL mass spectrometer operated in positive electrospray mode. Because of the very high number of compounds in both methods and the knowledge that future analogues are always immerging on the market that could thus be present in samples, a targeted/untargeted LC-MS/MS screening method was developed. For both projects, detection limits were in the sub ng/mL range and intra- and inter-assay precisions were below 10.5%. Recovery from real samples ranged from 92 to 111%. The innovation of the developed LC-MS/MS methods is that the full scan event in the MS acquisition provides accurate masses for all detected species and thus allows post-analysis identification of initially untargeted compounds, i.e., the immerging analogues. This innovation adds an additional dimension to traditional MS/MS methods, allowing high mass resolution of untargeted compounds.

Compared to males in almost any social group in all affluent nations, Australia's Aboriginal and Torres Strait Islander men suffer from substantially more serious illnesses and early death. To date, research done by or in collaboration with Indigenous communities has revealed the extent of the problems that arise from diabetes, heart disease, hypertension, cancers, respiratory diseases, psychological disorders, accidental injuries, violence and other causes. Reproductive health, however, rarely has been studied among Indigenous men. To date, research in this field has been limited mainly to studies of sexually transmitted infections. No data has been published on Aboriginal men's symptoms of prostate disease or erectile dysfunction, nor has the clinical screening and treatment of these disorders among these men been assessed. In-depth search of the worldwide web demonstrated that little information on these issues was available from other Indigenous populations. It does appear that Indigenous men in Australia, New Zealand and North America are less likely than European-ancestry men to die from prostate cancer, or for living cases to be recorded on cancer registries. This may arise because Indigenous men genuinely have a lower risk, or because they are not captured by official statistics, or because they do not live long enough to develop severe prostate disease. We also know very little about other reproductive health problems such as sexual dysfunction and specifically erectile difficulties. One reason for our scant knowledge is that research mainly relies on self-report of sensitive information. The aim of the research study was to improve the understanding of sexual and reproductive health problems experienced by Indigenous men. This is best gathered by Aboriginal males who are inside the culture of middleaged and older Indigenous men, but until now this has not been attempted. In this study we adopted the World Health Organization (WHO) definitions for Reproductive and Sexual Health (WHO, 2001). Thus, we consider reproductive system disorders (prostate disease, erectile dysfunction) and related health care-seeking, and also men's perceptions about a "satisfying and safe sexual life". The methodology was framed within an Aboriginal and Torres Strait Islander research protocol that advocates respect for cultural, social and community customs. A mixed method design combined qualitative inquiry (4 focus groups and 18 in-depth interviews) and quantitative survey (n=301) involving men living in remote, rural and urban communities (Tiwi Islands, Darwin and north and south-east Queensland). Survey data were compared to recently published self-reports from 5990 randomly selected men aged over 40 years in Australia (Holden et al., 2005, The Lancet, 366, 218-224. The qualitative interviews revealed that most men were silent about reproductive health. They were unwilling to reveal their inner feelings to wives or partners, and they were unwilling to discuss such issues with doctors and other health care workers. Men's reaction to sexual difficulties included shame, denial, substance abuse and occasionally violence. On a positive note many men said they want to learn about it, so they understand how to cope with such problems. The Indigenous men reported symptoms of erectile dysfunction at least as much as non-Indigenous men in other Australian studies. Bivariate analysis showed that erectile dysfunction was correlated with many health and lifestyle variable. However multivariate analysis revealed that only three factors significantly predicted ED: presence of chronic disease, presence of pain when working, and living in a remote geographic location The quantitative survey data indicate that Indigenous men have more symptoms of prostate disease than non-Indigenous men. The syndrome appears to be poorly managed in clinical practice (e.g. rates of PSA testing and digital-rectal examination are only one-third the rate reported by non-Aboriginal men, despite equivalent likelihood of GP visits). The research study adds to the literature by providing better insight and depth into the issues impacting on Aboriginal and Torres Strait Islander males experiencing reproductive and sexual health difficulties. It also provides a platform to undertake comprehensive research with Aboriginal and Torres Strait Islander men to explore a wider spectrum of questions in this important but neglected area. Implications for education of primary healthcare workers and community-based awareness campaigns for Indigenous males are discussed. Most of all, this study revealed "layers" of silence around sexual and reproductive health of Indigenous men. This includes silence in the scientific establishments in health services, and in the community. It is hoped that this study puts the voices of the men forward to help to break down this silence.