Nitric oxide is released from the endothelium and causes relaxation of vascular smooth muscle by stimulating guanylate cyclase to produce guanosine 3’,5’-cyclic monophosphate (cGMP) which, in turn, is degraded by phosphodiesterase type 5 (PDE5). Inhibition of PDE5, with drugs like sildenafil citrate, promotes NOstimulated relaxation of vascular smooth muscle. The overall aim of the work contained within this thesis was to further characterise the systemic vascular effects of PDE5 inhibition. Four clinical studies were performed. The aims of the first study were to investigate in healthy men the effect of smoking on endothelium-dependent vasomotor function measured as the change in peripheral arterial wave reflection with inhaled salbutamol, and the effect of acute sildenafil 100 mg on this response. Smokers (n=12) exhibited a reduced response to inhaled salbutamol compared to non-smokers (n=11) [mean(standard deviation) area under the curve of the change in central augmentation index following salbutamol 400 μg: -29(143) AU in smokers vs -159(124) AU in non-smokers, P=0.03]. In the smokers, there was a trend to an improvement in the response to salbutamol following sildenafil [-96(266) AU vs -29(143) AU with matched placebo; P=0.2]. The co-administration of glyceryl trinitrate (GTN) and sildenafil is absolutely contraindicated because of the potential for profound hypotension. The aim of the second study was to characterise the time course of this interaction. Twenty men with stable angina, maintained on their usual medicines, were administered sublingual GTN 400 μg 1, 4, 6 and 8 hours after sildenafil 100 mg or matched placebo. Compared to the combination of GTN and placebo, the combination of GTN and sildenafil resulted in greater mean maximum reductions from baseline in sitting systolic blood pressure (BP) at 1, 4 and 8 hours, and in sitting diastolic BP at all time points (all P<0.05). Compared to placebo, sildenafil alone reduced systolic BP at 1, 4, 6 and 8 hours (P<0.01 at 1 hour and P<0.05 at 4, 6, and 8 hours) and diastolic BP at 4, 6, and 8 hours (all P<0.01). Analysis of the change in BP from the measures taken before each GTN challenge suggested that the interaction on BP might be synergistic at 1 hour after sildenafil, but no more than additive at 6 and 8 hours after sildenafil. Symptoms consistent with hypotension occurred following GTN in 6 subjects at 1 hour and 3 subjects at 4 hours after sildenafil, but in no subjects at 6 and 8 hours after sildenafil or at any time after placebo. In the third study, 25 otherwise untreated hypertensives were given sildenafil 50 mg or matched placebo three times daily for 16 days and the effects on ambulatory BP, clinic BP, arterial wave reflection, carotid-femoral pulse wave velocity and brachial artery flow-mediated dilatation were measured. Three subjects were withdrawn because of side effects and the data from the remaining 22 subjects were analysed. Sildenafil reduced ambulatory BP [change from baseline in average daytime BP: systolic -8(9) mmHg vs 2(9) mmHg with placebo, P<0.01; diastolic -6(5) mmHg vs 0(6) mmHg, P<0.01] and clinic BP [change from baseline to 1 hour after drug administration on day 16: systolic -5(11) mmHg vs 4(10) mmHg, P<0.01; diastolic -5(5) mmHg vs 2(7) mmHg, P<0.01]. Sildenafil, but not placebo, reduced arterial wave reflection [central augmentation index from 32(9)% at baseline to 30(10)% at 1 hour after administration on day 16, P<0.05; radial augmentation index from 88(13)% to 84(13)%, P<0.01], but the change in arterial wave reflection was not statistically significant compared to the change with placebo. Sildenafil did not affect pulse wave velocity or flow-mediated dilatation. The fourth study investigated the potential of combined PDE5 inhibition and organic nitrate for the management of treatment-resistant hypertension (TRH). In 6 patients with TRH, maintained on their usual antihypertensives sildenafil 50 mg alone, isosorbide mononitrate (ISMN) 10 mg alone and co-administered sildenafil and ISMN all acutely reduced systolic BP and diastolic BP compared to placebo (quantified as the area under the curve of the change from baseline to 4 hours after drug administration; all P≤0.01). The combination produced a greater reduction in systolic BP than did either sildenafil alone (P=0.03) or ISMN alone (P=0.01) and a greater reduction in diastolic BP than did sildenafil alone (P=0.02). Compared to placebo, from 1 to 3 hours after drug administration BP was on average 13/10 mmHg lower with sildenafil alone, 18/14 mmHg lower with ISMN alone and 26/18 mmHg lower with the combination. The following conclusions were made. (1) Smokers exhibit impaired vascular responsiveness to inhaled salbutamol, indicating systemic endothelial dysfunction, which may be improved by sildenafil. (2) In men with stable angina there is an interaction on BP reduction between sildenafil 100 mg and sublingual GTN 400 μg for at least 8 hours after sildenafil administration, but this interaction is no more than additive from 6 hours after sildenafil administration. (3) Regular sildenafil monotherapy reduces BP in hypertension. (4) In patients with TRH maintained on their usual antihypertensives sildenafil alone and ISMN alone both acutely reduce BP and there is additional BP reduction when these drugs are given in combination.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-14T12:01:58Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Fabiana corrigida.pdf: 10988390 bytes, checksum: 0f7ed97ccd1c58e803213e43db1e6a2c (MD5) / Made available in DSpace on 2016-07-14T12:01:58Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Fabiana corrigida.pdf: 10988390 bytes, checksum: 0f7ed97ccd1c58e803213e43db1e6a2c (MD5) Previous issue date: 2015-07-29 / FACEPE / O Sildenafil é um inibidor potente e seletivo da fosfodiesterase-5 (PDE5). Este fármaco foi aprovado para uso terapêutico na disfunção erétil e, atualmente, vem sendo usado também no tratamento da hipertensão pulmonar. Embora mantenha um excelente nível de segurança e perfil de tolerabilidade, poucos estudos avaliaram os possíveis efeitos colaterais do tratamento crônico com Sildenafil sobre o sistema reprodutor masculino, especialmente na próstata visando o relaxamento da uretra e alívio dos Sintomas do Trato Urinário Inferior (STUI). Desta forma, avaliamos o efeito do tratamento, em camundongos C57Bl/6, através de análise morfológica, ultraestrutural e expressão molecular de guanilato ciclase solúvel-sGC, Óxido nítrico sintase endotelial-eNOS, Antígeno prostático específico-PSA e Fator de crescimento transformador beta-TGF- no tecido prostático. Foi-se observado que o tratamento com Sildenafil não induz danos evidentes na próstata. Além disso, tem sido demonstrado que Sildenafil tem eficácia terapêutica em doenças inflamatórias crônicas, podendo apresentar uma eficácia terapêutica potencial em diferentes doenças. Entre elas, uma atenção especial tem sido dada para as patologias relacionadas ao trato urogenital masculino, como a Hiperplasia Prostática Benigna (HPB), Câncer de próstata e Prostatites. A inflamação tem sido considerada como um fator etiológio da HPB e STUI. Assim, foi proposto um modelo de lesão prostática com injeção intrauretral de LPS (1mg/ml), em camundongos machos Swiss e C57Bl/6, desenvolvido durante 3, 7, 10 e 14 dias. A análise dos resultados mostrou que a indução intrauretral com lipopolisacarideo-LPS atua como importante agente da HPB, além de promover o aumento de fatores de crescimento (FGF-7 e FGF- β), α-actina e citocinas pró-inflamatórias (IL-1, IL-6, IL-17), tanto no estroma como no epitélio. Uma vez que o Sildenafil tem potencial anti-inflamatório, este estudo se propôs a analisar a ação do Sildenafil em um modelo de lesão prostática experimental, induzido por injeção intrauretral de LPS em camundongos C57BL/6. O tratamento com Sildenafil (25mg/kg) dos animais com prostatite apresentaram redução significativa de -actina, COX-2, NFK- B, IL-6, IL-17 e FGF-7. Por não induzir danos na próstata , o Sildenafil, por não induzir a longo prazo danos evidentes na próstata pode representar uma estratégia farmacológica para o tratamento de doenças inflamatórias crônicas do trato urogenital. / Sildenafil is a potent and selective inhibitor of phosphodiesterase-5 (PDE5). This drug has been approved for therapeutic use in erectile dysfunction and currently has been also used to treat pulmonary hypertension. While maintaining an excellent level of safety and tolerability profile, few studies have evaluated the possible side effects of chronic treatment with Sildenafil on the male reproductive system, especially in prostate aimed at relaxing the urethra and relief of symptoms of Lower Urinary Tract (LUTS). Therefore, we assessed the treatment effect in C57Bl/6 mice, using morphological analysis, ultrastructural and molecular expression of soluble guanylate cyclase-sGC, endothelial nitric oxide synthase, eNOS, prostate-specific antigen PSA and transforming growth factor beta TGF- in prostate tissue. It is observed that the treatment with sildenafil does not induce apparent damage to the prostate. Furthermore, Sildenafil has been shown to have therapeutic efficacy in chronic inflammatory diseases, which have a potential therapeutic efficacy in various diseases. Among them, special attention has been given to the pathologies related to the male urogenital tract, such as Benign Prostatic Hyperplasia (BPH), prostate cancer and Prostatitis. Inflammation has been considered as a factor etiológio of BPH and LUTS. Thus, it has been proposed a prostatic intraurethral injection injury model of LPS (1mg/ml) in male Swiss mice and C57Bl /6 developed for 3, 7, 10 and 14 days. The results showed that the intraurethral induction with lipopolysaccharide-LPS acts as an important agent of HPB, and to promote the increase of growth factors (FGF-7 and FGF- ), -actin and proinflammatory cytokines (IL- 1, IL-6, IL-17), both in the stroma and the epithelium. Since Sildenafil has potential anti-inflammatory, this study aimed to analyze the action of Sildenafil in an experimental prostate injury model induced by intraurethral injection of LPS in C57Bl/6 mice. Treatment with sildenafil (25 mg/kg) of animals with prostatitis showed a significant reduction of -actin, COX-2 NFK-kB, IL-6, IL-17 and FGF-7. By not induce damage to the prostate, Sildenafil, not to induce long term damage evident in the prostate may represent a pharmacologic strategy for the treatment of chronic inflammatory diseases of the urogenital tract.

Les antidépresseurs ont un effet antiallodynique qui dépend de la stimulation des récepteurs β2-adrénergiques. Ceux-ci stimulent la production d’adénosine monophosphate cyclique (AMPc) régulé par les phosphodiestérases de type 4 (PDE4). Nous avons ici étudié l’effet d’inhibiteurs de PDE (iPDE) sur la douleur neuropathique, grâce à des approches de pharmacologie comportementale chez la souris complétées par de l’imagerie calcium et des approches moléculaires. Nos résultats montrent un effet antiallodynique des iPDE4 et des iPDE5. L’action des iPDE4 est liée à une diminution d’expression du TNFα dans le ganglion rachidien et au recrutement des récepteurs delta des opioïdes. Celle des iPDE5 nécessite à la fois les récepteurs mu et delta. Nous montrons aussi que l’action d’un iPDE4 dépend de la dose, l’activation de cellules gliales semblant corrélée à l’effet antiallodynique à faible dose, alors que celle des neurones à forte dose a un effet pronociceptif via les récepteurs TRPV1. / Antidepressants have an antiallodynic action that is dependent on β2-adrenoceptor stimulation. These receptors stimulate the cAMP production, which is regulated by type 4 phosphodiesterases (PDE4). Here, we studied that action of PDE inhibitors (iPDE) on neuropathic pain, using behavioral pharmacology approaches in mice, completed by calcium imaging and molecular approaches. Our results show the iPDE4s and iPDE5s have an antiallodynic action. The iPDE4s act through a decreased expression of TNFα in dorsal root ganglia and the recruitment of the delta opioid receptors. The action of iPDE5 requires both mu and delta opioid receptors. We also show that the action of an iPDE4 depends on the dose, the activation of glial cells at low dose being correlated with an antiallodynic action, while the recruitment of neurons at higher doses has a pronociceptive action via TRPV1 receptors.

The cancers of liver, colon and breast are amongst the top five most prevalent and most fatal worldwide. As the Raf/MEK/ERK pathway is frequently deregulated in hepatocellular carcinoma (HCC), sorafenib, a Raf kinase inhibitor, became the first systemic therapy approved for the treatment of patients with HCC. However, sorafenib only produced modest effects with low response rates in the clinic. Similarly, regorafenib, which was approved for the treatment of metastatic colorectal cancer (CRC), has had a poor response rate in the clinic. Since phosphodiesterase type 5 has been reported to be overexpressed in HCC and CRC, we hypothesized that sildenafil, a phosphodiesterase type 5 inhibitor, could enhance the toxicities of sorafenib and regorafenib in HCC and CRC cells, respectively. Our in vitro data indicated that the drugs interacted strongly to kill cancer cells via induction of ER stress, autophagy and apoptosis. In accordance with these findings, our in vivo data demonstrated a significant reduction in tumor growth. The second study in this manuscript was conducted based on the growing body of evidence about the significant contribution of EGFR and JAK/STAT signaling to the breast tumorigenesis. Our preliminary in vitro data demonstrated that the concurrent inhibition of these two pathways by lapatinib, a dual ERBB1/2 inhibitor, and ruxolitinib, a JAK1/2 inhibitor, synergistically killed breast cancer cells of all types, including the resistant triple negative subtype. Our mechanistic studies showed that the combination of ruxolitinib and lapatinib triggered cytotoxic mitophagy, and autophagy-dependent activation of BAX and BAK leading to the mitochondrial dysfunction.

Orientador: Gilberto De Nucci / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T19:42:28Z (GMT). No. of bitstreams: 1 SantosFilho_HiltonOliveirados_D.pdf: 1622141 bytes, checksum: 5f817cf4a04ee643bdf7e261e5861691 (MD5) Previous issue date: 2013 / Resumo: Carbonato de Lodenafila é um novo tipo de inibidor de fosfodiesterase 5 (PDE5) utilizado no tratamento da disfunção erétil. O presente estudo foi realizado para avaliar a segurança, tolerabilidade e farmacocinética do carbonato de lodenafila após a administração de doses orais únicas ascendentes (de 1 a 100 mg) a voluntários saudáveis do sexo masculino (n = 33). O estudo foi um estudo clínico fase I, aberto, de escalonamento de dose, utilizando a administração de doses orais únicas de carbonato de lodenafila. Carbonato de Lodenafila foi administrado sequencialmente escalonado em doses únicas de 1 mg a 100 mg com um período sem uso do medicamento (washout) de pelo menos 1 semana, entre cada dose. A progressão para a próxima dose foi permitida se após a avaliação dos exames clínicos, laboratoriais e Monitorização Ambulatorial da Pressão Arterial (MAPA), não demonstrassem alterações e eventos adversos sem relevância clínica. As amostras de sangue foram coletadas na pré-dose e em intervalos determinados e 24 horas após a administração. As amostras de plasma para a mensuração de carbonato lodenafila e lodenafila foram analisadas por cromatografia líquida acoplada à espectrometria de massa. Não foram observados eventos adversos graves, e nenhum dos voluntários abandonou o estudo devido à intolerância. As medições do MAPA, exames clínicos e laboratoriais e ECG não revelaram alterações significativas mesmo em doses mais elevadas. Carbonato Lodenafila não foi detectado em qualquer amostra, indicando que ele atua como um pró-droga. Os parâmetros farmacocinéticos médios de lodenafila para tmax e t1 / 2 foram 1,6 (± 0,4) horas e 3,3 (± 1,1) horas, respectivamente. Este estudo demonstrou que o carbonato de lodenafila é bem tolerado e apresentou um bom perfil de segurança em voluntários saudáveis do sexo masculino / Abstract: Lodenafil carbonate is a new phosphodiesterase type 5 (PDE5) inhibitor used in treatment of erectile dysfunction. The present study was conducted to evaluate the safety, tolerability, and pharmacokinetics of lodenafil carbonate after administering ascending (1 to 100 mg) single oral doses to healthy male volunteers (n=33). The study was an open-label, dose-escalation, phase I clinical trial involving the administration of single oral doses of lodenafil carbonate. Lodenafil carbonate was administered sequentially, escalating in single doses of 1 mg to 100 mg with a washout period of at least 1 week between each dose. The progression to the next dose was allowed after clinical and laboratory exams, Ambulatory Monitoring of Arterial Pressure (AMAP) without relevant clinical modifications and adverse events without clinical relevancy. Blood samples were collected at pre-dose, determined intervals and 24h postdosing. Plasma samples for measurement of lodenafil carbonate and lodenafil were analyzed by liquid chromatography coupled to tandem mass spectrometry. No serious adverse events were observed, and none of the subjects discontinued the study due to intolerance. The AMAP measurements, clinical and laboratory exams and ECG revealed no significant changes even at higher doses. Lodenafil carbonate was not detected in any samples, indicating that it acts as a pro-drug. The mean lodenafil pharmacokinetic parameters for tmax and t1/2 were 1.6 (±0.4) hr and 3.3 (±1.1) hr, respectively. This study demonstrated that lodenafil carbonate was well tolerated and showed a good safety profile in healthy male volunteers / Doutorado / Clinica Medica / Doutor em Clínica Médica

Endothelial cells (EC) are essential regulator of vascular homeostasis through the generation and release of various bioactive agents, including nitric oxide (NO). NO modulates several vascular functions such as vascular tone and permeability, through the stimulation of soluble guanylate cyclase (sGC) leading to the production of cGMP. Conversely, phosphodiesterases (PDEs) are enzymes metabolizing cyclic nucleotides (cGMP and cAMP) and are therefore major regulatory players for cGMP and cAMP signalling pathways. Although ECs are the main source of NO, little is known on the endothelial NO-cGMP signalling pathway and cellular outcomes. It was then hypothesized that a specific population of cGMP-phosphodiesterases allows ECs to stabilize cGMP levels despite the elevated production of NO. Expression of cGMP-phosphodiesterases was initially studied in resistance mesenteric arteries from mice. PDE5 and PDE6 were both found at mRNA and protein levels in native arteries but PDE6 is not found in cultured ECs. Interestingly, subcellular distributions of both enzymes were distinct. PDE5 appeared to be homogeneously distributed whilst PDE6 catalytic subunits (PDE6 and PDE6) showed a preferential staining in the perinuclear region. These results suggest that PDE6 might be involved in the regulation of cGMP microdomains. Based on these findings, a mathematical model was developed. Simulations of dynamic cGMP levels in ECs support the notion of cGMP microdomains dependent on PDE6 expression and localization. In the absence of PDE6, application of NO either as a single bolus or repetitive pulses led to a homogeneous increase in cGMP levels in ECs despite PDE5 homogeneous distribution. However, PDE6 subcellular targeting to the perinuclear membrane generated a cGMP-depleted perinuclear space. The findings from this study provide the first evidence of the expression and specific intracellular distribution of PDE6 in native endothelial cells that strongly support their involvement in the generation of cGMP microdomains / Les cellules endothéliales (CEs) participent au maintien de l’homéostasie vasculaire en générant et libérant de nombreux agents bioactifs, incluant l’oxyde nitrique (NO). Le NO module plusieurs fonctions vasculaires telles que le tonus et la perméabilité vasculaire via la stimulation de la guanylate cyclase soluble (GCs) provoquant la formation de GMPc. D’autre part, les phosphodiestérases (PDEs) sont des enzymes métabolisant les nucléotides cycliques (GMPc et AMPc) et participent donc à des étapes essentielles du contrôle des voies de signalisation du GMPc et de l’AMPc. Bien que les CEs soient la source principale de NO, la voie de signalisation NO-GMPc endothéliale et les répercussions fonctionnelles demeurent méconnues. Nous avons alors émis l’hypothèse qu’une population spécifique de PDEs ciblant le GMPc (PDEs-GMPc) permettrait aux CEs de maintenir des niveaux de GMPc faible malgré l’importante production de NO. L’expression des isoformes de PDEs-GMPc dans les artères mésentériques de souris fut initialement déterminée. PDE5 et PDE6 furent détectées tant sous la forme d’ARNm que de protéines dans les artères natives alors que PDE6 est absente de lignées de CEs en culture. La distribution intracellulaire des deux enzymes est distincte. Alors que PDE5 est distribué uniformément dans le cytoplasme des cellules endothéliales, les sousunités catalytiques de PDE6 ( et ) sont préférentiellement présentes dans la région périnucléaire. Ces résultats suggèrent que PDE6 puisse être impliqué dans le contrôle de microdomaines de GMPc. Des simulations effectuées à l’aide d’un modèle mathématique développé sur la base de ces données sont en accords avec la notion selon laquelle l’expression et la distribution subcellulaire de PDE6 sont responsables de microdomaines de GMPc dans l’endothélium. En absence de PDE6, l’ajout de NO sous forme de bolus unique ou répétée mène à une augmentation homogène de la concentration cytoplasmique en GMPc malgré la présence de PDE5. Toutefois, la présence de PDE6 à la membrane péri-nucléaire crée un espace péri-nucléaire pauvre en GMPc. Les résultats de cette étude forment les premières évidences de l’expression et de la distribution intracellulaire hétérogène de PDE6 dans les cellules endothéliales natives et suggèrent leur implication dans la génération de microdomaines.

Les douleurs neuropathiques désignent une hypersensibilité du système nerveux central sensoriel. C’est une maladie chronique et handicapante qui touche environ 6% de la population française. Cependant, à l’heure actuelle, il n’existe pas de traitement spécifique et efficace. Dans le cadre de cette thèse, nous avons utilisé deux stratégies différentes afin de développer des outils pharmacologiques originaux pour traiter ces douleurs : une approche phénotypique autour de deux inhibiteurs de la surproduction de TNFα (un dérivé de 2-aminopyrimidine et un dérivé de pyridin-2-yl guanidine) et une approche moléculaire autour du MY 5445 (un inhibiteur de PDE5 dérivé de phtalazine). En particulier, notre travail s’est basé sur la conception, la synthèse et l’étude des relations structure-activité autour de ces différents hits et nous avons obtenu des composés efficaces par voie i.p. ou per-os sur un modèle murin de douleurs neuropathiques.En parallèle de ce travail de pharmacologie, nous avons développé différents systèmes catalytiques (Pd, Cu) en milieu micellaire afin de former des liaisons C-N à température quasi-ambiante. / Neuropathic pains correspond to a central sensory nervous system hypersensitivity. It is a chronic and disabling disease, which touch around 6% of the French population. Nowadays, there is no specific and efficient treatment. In my PhD project, we used two different strategies in order to develop innovative pharmacological tools to treat those pains: a phenotypic approach around two TNFα overproduction inhibitor (a 2-aminopyrimidine derivative and a pyridin-2-yl guanidine derivative) and a molecular approach around MY 5445 (a phthalazine PDE5 inhibitor). Our work was based on the design, synthesis and structure-activity relationship study around various hits and we obtained compounds i.p. and orally effective on a murin neuropathic pain model.In parallel to this pharmacological work, we developed different catalytic systems (Pd, Cu) under micellar conditions to form C-N bonds at almost room temperature.