Purpose: To establish a tool, termed as antimuscarinic activity (AMA), to predict the incidence of antimuscarinic adverse events (AMAEs).Methods: A literature review, focused on drugs having off-target interaction with muscarinic receptors, was performed. Prototypical drugs olanzapine, diphenhydramine, paroxetine were selected for the analysis. Scopolamine and darifenacin were included as positive and negative controls, respectively. Physiochemical properties, pharmacokinetic data, and clinical incidence of AMAEs for the selected drugs were collected from reported literature. Extrapolation of literature data was carried-out to obtain exposure data. To determine the drugs muscarinic affinity (Ki values), experiments were performed using 3H-QNB and membrane suspensions of M1, M2, and M3. Cmax, values were combined with Ki values to generate the relevant AMA. Validation of the AMA biomarker was carried-out against the reported AMAEs incidence. Results: With the exclusion of scopolamine and olanzapine for CNS and peripheral AMAEs, respectively, AMA ranking was related to the drugs AMAEs.

This thesis deals with effects of some chosen 7-methoxitacrine (7-MEOTA) analogues on enzymatic activity of acetylcholinesterase (AChE). 7-MEOTA is a derivative of tacrine, which had been used for symptomatic treatment of Alzheimer's disease (AD), until drugs with better therapeutic index were developed. 7-MEOTA the same way as tacrine therapeutically acts by inhibition of acetylcholinesterase and a neurotransmitter acetylcholine rise in the organism. It shows similar strength and type of inhibition, but it's less toxic contrary to tacrine. Some of the previously examined analogues of 7-MEOTA were as strong or even stronger AChE inhibitors than 7-MEOTA and so promising future medicaments. However, all the compounds analyzed in this thesis showed weaker enzymatic reaction inhibition and AChE affinity. For each of the examined compounds IC50, Ki and Ki' were calculated and AChE inhibition type was determined. All the 7-MEOTA analogues showed a mixed type of the inhibition. The theoretical part of this thesis deals with manifestations and origins of AD, its genetic factors etc. and tries to show some of the anthropological findings a theories connected with the theme.

This thesis deals with effects of some chosen 7-methoxitacrine (7 MEOTA) analogues on enzymatic activity of acetylcholinesterase (AChE). 7-MEOTA is a derivative of tacrine, which had been used for symptomatic treatment of Alzheimer's disease (AD), until drugs with better therapeutic index were developed. 7-MEOTA the same way as tacrine therapeutically acts by inhibition of acetylcholinesterase and a neurotransmitter acetylcholine rise in the organism. It shows similar strength and type of inhibition, but it's less toxic contrary to tacrine. Some of the previously examined analogues of 7-MEOTA were as strong or even stronger AChE inhibitors than 7-MEOTA and so promising future medicaments. However, all the compounds analyzed in this thesis showed weaker enzymatic reaction inhibition and AChE affinity. For each of the examined compounds IC50, Ki and Ki' were calculated and AChE inhibition type was determined. All the 7 MEOTA analogues showed a mixed type of the inhibition. The theoretical part of this thesis deals with manifestations and origins of AD, its genetic factors etc. and tries to show some of the anthropological findings a theories connected with the theme. Powered by TCPDF (www.tcpdf.org)

Our project was aimed at the development of novel CB1 cannabinoid receptor antagonists that may have clinical applications for the treatment of cannabinoid and psychostimulant addiction. In this study, we designed, synthesized, and established the CB1 affinity for the 1,5-diaryl-1,2,3- triazole esters, a series of 4,5-diaryl-1-substituted-1,2,3-triazole analogues and a series of 4,5- diaryl-2-substituted-1,2,3-triazoles. Our research group has been interested in the synthesis of amphibian alkaloids due to their interesting biological activities. We have recently developed a general synthetic strategy which can rapidly prepare a few amphibian alkaloids simply from the abundant natural product (-)- cocaine This strategy was first successfully applied to the synthesis of (-)-monomorine. More recently, this strategy has also been utilized in the syntheses of both of the enantiomers of cispyrrolidine 225H and (+)-gephyrotoxin 287C.

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative dont les premiers symptômes apparaissent généralement vers 60 ans. Elle affecte sélectivement le système moteur et provoque une paralysie progressive amenant au décès du patient par défaillance respiratoire en quelques années. À ce jour, il n’existe aucun traitement curatif, d’où la nécessité de comprendre la physiopathologie de la SLA. Bien que de nombreuses altérations dans le muscle aient été mises en évidence, sa contribution dans la SLA reste à définir. Nous avons montré que FUS est enrichi dans les noyaux sous-synaptiques de façon dépendante de l’innervation. De plus, FUS se lie au promoteur des récepteurs de l’acétylcholine et induit leur transcription de façon dépendante d’ERM. Le mutant FUS, quant à lui, est enrichi dans les noyaux extra-synaptiques et entraîne une toxicité musculaire responsable de l’altération de la jonction neuromusculaire (JNM). Au-delà de la JNM, FUS active MEF2A, de façon dépendante de PRMT1 afin de réguler les fonctions mitochondriales et la différenciation musculaire. La toxicité musculaire de FUS joue donc un rôle clé dans la physiopathologie de la SLA. / Amyotrophic lateral sclerosis is a neurodegenerative whose first symptoms generally appear around age 60. It is characterized by progressive motor neuron degeneration, paralysis and leading to death due to respiratory failure in a few years. Currently, there is no cure so the understanding of ALS physiopathology is necessary. Although many alterations in the muscle have been highlighted, its contribution in ALS remains to be defined. We showed that FUS is enriched in subsynaptic nuclei and this enrichment depended on innervation. Besides, FUS binds directly acetylcholine receptors (AchR) promoter and is required for Ermdependent induction of AChR expression. Conversely, mutant FUS is enriched on extra-synaptic nuclei and induce muscle intrinsic toxicity responsible for neuromuscular junction (NMJ) alteration. Beyond NMJ, FUS is required for muscle mitochondrial function and muscle differentiation through PRMT1-dependent MEF2A activation. Thus, FUS muscular toxicity plays a key role in the ALS physiopathology.

Estudos anteriores de nosso laboratório demonstraram que a estimulação aguda dos receptores 2-adrenérgicos (2-AR) atenua a perda de massa muscular induzida pela desnervação motora (DEN) por meio de uma via dependente de AMPc/PKA. No entanto os mecanismos moleculares envolvidos na ativação crônica destes receptores ainda são pouco conhecidos. Por outro lado, a ativação desta via de sinalização também está envolvida no controle da estabilidade dos receptores nicotínicos (AChR) na junção neuromuscular (JNM), sugerindo que a densidade dos AChR possa estar sob controle neuro-humoral. Desta forma, aventou-se a possibilidade de que além dos efeitos protetores na massa muscular, a ativação dos receptores 2-AR pudesse mediar a estabilização dos AChR na placa motora. Para testar essa hipótese, camundongos foram submetidos à DEN através da secção do nervo ciático, um protocolo clássico de indução de atrofia muscular e desestabilização dos AChR, e tratados com salina ou clembuterol (CB), um 2-agonista seletivo, por até 14 dias. Após 3 dias de DEN, observou-se redução da massa muscular e aumento do conteúdo proteico e expressão do RNAm de genes relacionados à ativação do sistema Ubiquitina-Proteassoma (atrogina-1 e MuRF1) e do sistema autofágico/lisossomal (catepsina L e LC3). A DEN também promoveu aumento no turnover dos AChR, no número de vesículas endocíticas e na expressão do RNAm para a subunidade 1 dos AChR. Após 7 dias, a DEN reduziu a expressão dos genes relacionados à atrofia e aumentou a atividade da via do AMPc/PKA independentemente do tratamento com CB. Na tentativa de elucidar os sinais extracelulares que produziam esta resposta adaptativa, foi demonstrado que neurônios catecolaminérgicos trafegam ao longo do nervo ciático e sua ablação pela DEN reduziu o conteúdo de noradrenalina muscular. Baseados nestes resultados, foi postulado a existência de uma hipersenbilidade às catecolaminas em músculos desnervados cronicamente. O tratamento com CB por 3 dias aboliu o aumento da expressão dos atrogenes induzido pela DEN e este efeito foi associado ao maior conteúdo de AMPc e de substratos fosforilados pela PKA. Além disso, o CB diminuiu a hiperexpressão do RNAm para catepsina L e LC3 induzida pela DEN de 7 dias. Embora o CB não tenha alterado a meia-vida dos AChR em músculos inervados e desnervados, houve um total bloqueio do aumento do número de vesículas endocíticas contendo o AChR em músculos desnervados e tratados com CB. Corroborando estes dados, o CB aumentou a incorporação de AChR novos nas JNM e este efeito foi também associado à maior expressão do RNAm para a subunidade 1-AChR em músculos desnervados. Esta ação do CB no turnover dos AChR parece ser direta uma vez que neuroniôs catecolaminérgicos presentes no nervo ciático ativam receptores 2-ARe a produção de AMPc especificamente na JNM. Em estudos in vitro, foi demonstrado que a estimulação colinérgica produzida pelo carbacol (10-4M) diminuiu a velocidade de síntese de proteínas, aumentou a proteólise total e a atividade do sistema proteolítico Ca2+-dependente em músculos soleus de ratos por meio da ativação dos receptores nicotínicos. Este efeito catabólico do carbacol foi completamente bloqueado pela adição de CB (10-4M) ao meio de incubação. Os dados obtidos no presente estudo permitem sugerir que a estimulação crônica dos 2-AR no músculo esquelético induz um efeito anti-catabólico pela supressão dos sistemas proteolíticos proteassomal e lisossomal, provavelmente através da via de sinalização do AMPc/PKA. A inibição destes sistemas pode estar relacionada ao aumento do turnover dos AChR, uma vez que a velocidade de incorporação destes receptores na JNM foi aumentada pelo CB. Além disso, os achados que mostram a associação entre neurônios noradrenérgicos e colinérgicos no nervo ciático, que conjuntamente inervam as JNM, e a co-localização de receptores 2-AR e AChR na sinapse permitem sugerir a existência de uma interação funcional entre o sistema colinérgico e adrenérgico na manutenção da massa muscular e da placa motora. / Previous studies from our laboratory have shown that the acute stimulation of 2-adrenergic receptor (2-AR) attenuates the muscle loss induced by motor denervation (DEN) through a cAMP/PKA dependent pathway. However, the molecular mechanisms involved in the chronic activation of these receptors are poorly understood. Furthermore, the activation of this signaling pathway is also involved in controlling the stability of nicotinic receptors (AChR) at the neuromuscular junction (NMJ), suggesting that the density of AChR may be under neurohumoral control. Thus, we postulated that besides the protective effects on muscle mass the activation of 2-AR receptors could mediate the stabilization of AChR in the motor plate. To test this hypothesis, mice were submitted to DEN through of the sciatic nerve section, a classical protocol of induction muscle atrophy and destabilization of AChR, and were treated with saline or clenbuterol (CB), a selective 2-agonist for 14 days. DEN decreased the muscle mass and increased the protein content and mRNA expression of genes related to the activation of the ubiquitin-proteasome system (atrogin-1 and MuRF1) and autophagic/lysosomal system (cathepsin L and LC3). DEN also promoted an increase in the turnover of AChR, number of endocytic vesicles and the expression of mRNA for the 1 subunit of AChR. Interestingly, chronic DEN induced down-regulation of atrophy related-genes, and increased the activity of cAMP/PKA pathway independently of CB treatment. In an attempt to elucidate the extracellular signals that produced this adaptive response, it was demonstrated that catecholaminergic neurons travels along the sciatic nerve and its ablation by DEN reduces muscle norepinephrine content. Based on these results, it was postulated the existence of a muscle adrenergic hypersensitivity to circulating catecholamines induced by chronic DEN. CB treatment for 3 days completely abolished the higher expression of atrogenes and this effect was associated with increased Camp content and PKA phosphorylated substrates. Furthermore, CB decreased the DEN-induced hyperexpression of cathepsin L and LC3 mRNA at 7 days. Although CB has not altered the half-life of AChR in innervated and denervated muscles, it produced a total blockage of the increased number of endocytic vesicles containing the AChR in denervated muscles. Consistently, CB increased the incorporation of new AChR and this effect was associated with an increased expression of the 1-subunit AChR mRNA in denervated muscles. This action of CB on AChR turnover appears to be direct, since catecholaminergic neurons are present in the sciatic nerve stimulating 2-AR and cAMP production specifically in the NMJ. Furthermore, in vitro studies demonstrated that cholinergic stimulation produced by carbachol (10-4M) decreased the rate of protein synthesis and increased the proteolytic activity of Ca2+-dependent system in rat soleus muscle through activation of nicotinic receptors. This catabolic effect of carbachol was completely blocked by the addition of CB (10-4M) to the incubation medium. These data suggest that chronic stimulation of 2-AR in skeletal muscle induces an anti-catabolic effect by suppressing proteasomal and lysosomal proteolytic systems, probably through the cAMP/PKA signaling. The inhibition of these systems seems to be related to the increased AChR incorporation into NMJ induced by CB treatment. Moreover, the association between noradrenergic and cholinergic neurons in the sciatic nerve, both of which innervate the motor endplates, and the co-localization of AChR and 2-ARat the synapse suggest the existence of a functional interaction between cholinergic and adrenergic systems in the maintenance of muscle mass and motor endplate.

Introdução: A hiperidrose primária (HP) é uma desordem que afeta negativamente a qualidade de vida de seus portadores. A fisiopatologia da HP não é bem compreendida e acredita-se que uma complexa disfunção do sistema nervoso simpático esteja relacionada com sua etiologia. A ressecção de um ou mais gânglios da cadeia simpática torácica constitui-se como o método mais eficiente de controle da HP; apesar disso, pouco se sabe sobre o funcionamento dos gânglios simpáticos em indivíduos normais e em portadores de HP. Objetivos: Analisar a expressão de acetilcolina e das subunidades 3 e 7 de seu receptor nicotínico neuronal em gânglios da cadeia simpática torácica de portadores de HP palmar e comparar estes resultados com os obtidos de não portadores; avaliar se existe diferença de tamanho entre esses gânglios. Métodos: Estudo transversal, no qual foram analisados dois grupos de 20 participantes: no grupo Hiperidrose, portadores de HP palmar, candidatos a simpatectomia torácica; no grupo Controle, doadores falecidos de órgãos sem história prévia de sudorese excessiva. Em todos os indivíduos foram realizados: ressecção do 3º gânglio simpático esquerdo; aferição do maior diâmetro do gânglio; avaliação imunohistoquímica pela quantificação das áreas de expressão forte e fraca de anticorpos primários contra acetilcolina e contra as subunidades 3 e 7 de seu receptor nicotínico neuronal. Resultados: A mediana da idade dos participantes foi menor no grupo Hiperidrose em relação ao Controle; a proporção de homens e mulheres foi de 3:17 no grupo Hiperidrose e 9:11 no Controle. A expressão da subunidade 3 foi semelhante em ambos os grupos (p = 0,78 para expressão forte e p = 0,31 para expressão fraca). A área de expressão forte da subunidade 7 correspondeu a 4,85% da área total em portadores de HP e a 2,34% nos controles (p < 0,001), enquanto a área de expressão fraca foi de 11,48% no grupo Hiperidrose e de 4,59% no Controle (p < 0,001). Expressão forte da acetilcolina foi encontrada em 4,95% da área total no grupo Hiperidrose e 1,19% no Controle (p < 0,001); expressão fraca foi encontrada em 18,55% e 6,77%, respectivamente (p < 0,001). O diâmetro dos gânglios ressecados foi de 0,71cm no grupo Hiperidrose e de 0,53cm no Controle (p < 0,001). Conclusões: Existe um aumento da expressão de acetilcolina e da subunidade 7 do seu receptor nos gânglios simpáticos de portadores de HP; a subunidade 3 do receptor nicotínico de acetilcolina tem expressão semelhante em gânglios simpáticos de portadores de HP e de não portadores; gânglios da cadeia simpática torácica apresentam diâmetro maior em portadores de HP / Introduction: Primary hyperhidrosis (PH) is a disorder that impairs the quality of life of its bearers. The PH physiopathology is not well understood and a complex sympathetic nervous system dysfunction seems to be related with its etiology. The resection of one or more thoracic sympathetic chain ganglia is the most effective PH treatment; however sympathetic ganglia function in normal subjects and in PH patients is unknown. Objectives: Analyzing the immunohistochemical expression of acetylcholine and its neuronal nicotinic receptors 3 and 7 subunits in thoracic sympathetic ganglia of PH patients and compare the results with those obtained from subjects without this disorder; identifying possible differences in size of these ganglia. Methods: Cross-sectional study, in which two groups of 20 subjects were analyzed: the Hyperhidrosis group, with palmar PH patients eligible to thoracic sympathectomy and the Control group, with organ donators after brain death without hyperhidrosis historical. For each subject it were performed: resection of the third left sympathetic ganglion; measurement of the ganglions diameter; immunohistochemical evaluation by quantification of intense and mild expression areas of primary antibodies against acetylcholine and its neuronal nicotinic receptors 3 and 7 subunits. Results: The median of participants age was smaller in Hyperhidrosis group than in Control; the male/female ratio was 3:17 in Hyperhidrosis group and 9:11 in Control. The 3 subunit expression was similar in both groups (p = 0.78 for intense expression and p = 0.31 for mild expression). Intense 7 subunit expression area was 4.85% in PH patients and 2.34% in controls (p < 0.001) whereas mild expression area was 11.48% in Hyperhidrosis group and 4.59% in Control (p < 0.001). Intense acetylcholine expression was found in 4.95% of total area in Hyperhidrosis group and in 1.19% in Control (p < 0.001); mild expression was found in 18.55% and 6.77%, respectively (p < 0.001). Ganglia diameter was 0.71cm in Hyperhidrosis group and 0.53cm in Control (p < 0.001). Conclusions: There is a higher expression of acetylcholine and its neuronal nicotinic receptors 7 subunit in sympathetic ganglia of PH patients; the 3 subunit of the neuronal nicotinic acetylcholine receptor shows similar expression in sympathetic ganglia of PH patients and subjects without this disorder; thoracic sympathetic chain ganglia diameter is bigger in PH patients

Nicotine has multiple behavioral effects as a result of its action in the central nervous system. Nicotine strengthens the behaviors that lead to nicotine administration (primary reinforcement), and this effect of nicotine depends on mesotelencephalic systems of the brain that are critical to goal directed behavior, reward, and reinforcement. Nicotine also serves as a ‘reinforcement enhancer’ – drug administration enhances behaviors that lead to other drug and nondrug reinforcers. Although the reinforcement enhancing effects of nicotine may promote tobacco use in the face of associated negative health outcomes, the neuroanatomical systems that mediate this effect of nicotine have never been described. The ventral tegmental area (VTA) is a nucleus that serves as a convergence point in the mesotelencephalic system, plays a substantial role in reinforcement by both drug and nondrug rewards and is rich in both presynaptic and postsynaptic nicotinic acetylcholine receptors (nAChRs). Therefore, these experiments were designed to determine the role of the VTA and nAChR subtypes in the reinforcement enhancing effect of nicotine. Transiently inhibiting the VTA with a gamma amino butyric acid (GABA) agonist cocktail (baclofen and muscimol) reduced both primary reinforcement by a visual stimulus and the reinforcement enhancing effect of nicotine, without producing nonspecific suppression of activity. Intra-VTA infusions of a high concentration of mecamylamine a nonselective nAChR antagonist, or methylycaconitine, an α7 nAChR antagonist, did not reduce the reinforcement enhancing effect of nicotine. Intra-VTA infusions of a low concentration of mecamylamine and dihydro-beta-erythroidine (DHβE), a selective antagonist of nAChRs containing the *β2 subunit, attenuated, but did not abolish, the reinforcement enhancing effect of nicotine. In follow-up tests replacing systemic nicotine injections with intra-VTA infusions (70mM, 105mM) resulted in complete substitution of the reinforcement enhancing effects – increases in operant responding were comparable to giving injections of systemic nicotine. These results suggest that *β2-subunit containing nAChRs in the VTA play a role in the reinforcement enhancing effect of nicotine. However, when nicotine is administered systemically these reinforcement enhancing effects may depend on the action of nicotine at nAChRs in multiple brain nuclei.

Alzheimer’s disease is a progressive neurodegenerative disorder generally affecting people above the age of 65 years. Even though the pathophysiological hallmarks of AD were established more than a hundred years ago, there is yet to be a drug that can stop its characteristic neuronal damage. Of the five currently FDA-approved drugs, galantamine has a unique mechanism of action. Apart from being an AChE inhibitor, galantamine can effectively potentiate (positive allosteric modulator) the effect of agonists at nAChRs at concentrations lower than those required for its action as an AChE inhibitor. Perhaps the clinical benefits observed with galantamine are associated mainly with its nAChRs-PAM action and not its AChE inhibitory effect. Inhibiting AChE causes a delay in the degradation of ACh and a prolonged presence of ACh might act at either nAChRs or mAChRs. By indirectly targeting mAChRs as well, AChE inhibitors may lead to potential side effects. Hence there is a need for specific nAChR agents. The aim of this study was to identify the structural features of galantamine that contribute solely towards its a7 nAChR-PAM effect. In doing so, we wish to divorce the structural features that might be important for interacting with AChE. Using the deconstruction approach, we have synthesized structurally abbreviated analogs of galantamine. To study the probable interactions, we docked these molecules in human a7 nAChR homology models. Ultimately, it is of interest to determine which analogs retain the PAM activity of galantamine and to address that, a preliminary screening was performed with a select few analogs using the two-electrode voltage clamp technique

<p>This thesis describes the design and synthesis of molecules with potential roles in biomedicine, with an emphasis on molecular recognition in complex biological environments. The first chapter describes the synthesis and evaluation of compounds for use in nuclide therapy. Carboranes are frequently used in the development of drugs for Boron Neutron Capture Therapy. New routes for monohydroxylation at the B and C atoms of <i>p</i>-carborane have been developed. The Suzuki-Miyaura reaction has been applied to the cross-coupling of <i>bis</i>(neopentyl glycolato)diboron or <i>bis</i>(pinacolato)diboron and 2-I-<i>p</i>-carborane. The synthesized derivatives are important intermediates in the synthesis of a number of potentially biologically active carborane-containing molecules.</p><p>The DNA intercalator doxorubicin has been functionalized to enable ¹²⁵I labelling. The aim of combining the DNA intercalator with ¹²⁵I was to achieve high delivery of cytotoxic radiation to the nucleus. The DNA-binding ability and cellular uptake of the synthesized compounds have been evaluated. One of the compounds bound strongly to DNA and had similar cellular uptake as daunorubicin, which makes the compound very interesting for further biological evaluation.</p><p>The second chapter describes the use of polypeptide conjugates to broaden our knowledge of molecular recognition. The polypeptides consist of 42 amino acids each and are designed to fold into helix-loop-helix motifs that dimerize due to their amphiphilic character. The polypeptides are combined with a variety of small organic molecules. The incorporation of small aromatic molecules to influence the structure and dynamics of a polypeptide has been investigated. By attaching a dansyl group to the side chain of a lysine residue, the dynamics of the protein’s hydrophobic core where affected to such a degree that a native-like fold was formed. The polypeptide conjugates have also been used to study the binding and recognition of native proteins. High-affinity binders for chitinases and acetylcholine esterase have been developed and evaluated.</p>