Synthese 18 F-markierter Liganden zur Visualisierung der GABA-Bindungsstelle des GABA A-Rezeptors mittels PET

γ-Aminobuttersäure (GABA) ist der wichtigste inhibitorische Neurotransmitter im zentralen Nervensystem und bindet vorrangig an ionotrope GABAA-Rezeptoren. Diese sind an fast allen neuronalen Prozessen beteiligt und werden darüber hinaus mit neurologischen Erkrankungen wie Epilepsie, Angstzuständen, Schlafstörungen und Schizophrenie in Verbindung gebracht. Die PET bietet als molekulares bildgebendes Verfahren die Möglichkeit einzelne Stoffwechselvorgänge des GABAergen Systems zu visualisieren und zu quantifizieren. Durch den Einsatz eines 18F-markierten Radioliganden an die GABA-Bindungsstelle könnten so die Rezeptorverfügbarkeit des GABAA-Rezeptors gemessen und die Ausschüttung des Neurotransmitters GABA quantifiziert werden.rn4-(2-Naphthylmethyl)-5-(piperidin-4-yl)isothiazolole und -isoxazolole stellen aufgrund ihrer hohen Affinität gegenüber der GABA-Bindungsstelle und ihrer lipophilen Struktur vielversprechende Leitstrukturen für die Entwicklung eines PET-Tracers zur Visualisierung der GABA-Bindungsstelle dar. Daher wurden zunächst 19F-substituierte Referenzverbindungen synthetisiert, um diese hinsichtlich ihrer Eignung als Radioligand in in vitro-Studien zu evaluieren. Dazu wurde Fluor direkt sowie über eine Fluorethoxygruppe an Position 1 des Naphthalinrings eingeführt. Zusätzlich wurde ein Fluorethylether eines Isothiazolols als Referenz-verbindung synthetisiert. In anschließenden Verdrängungsstudien wurden die Affinitäten der synthetisierten Verbindungen mit [3H]Muscimol an Membranpräparaten aus Rattenhirnen, sowie transfizierten HEK293-Zellen bestimmt. Zusätzlich wurden die entsprechenden Log D-Werte bestimmt. Die Verbindung 5-(piperidin-4-yl)-4-(1-fluornaphth-2-ylmethyl)-isothiazol-3-ol VK5 zeigte in den in vitro-Studien die vielversprechendsten Ergebnisse (IC50 = 10 nM; Log D = 1,7) und wurde im Folgenden in einer dreistufigen Radiosynthese als 18F-Verbindung synthetisiert.rnZu diesem Zweck wurde ein geeigneter Markierungsvorläufer dargestellt und über eine n.c.a. SNAr-Markierung mit [18F]F- umgesetzt. Die Reaktionsparameter wurden hinsichtlich Reaktionszeit, -temperatur, Basenkonzentration und Lösungsmittel optimiert. Die zur Aktivierung einer SNAr ein-geführte Carbonylfunktion wurde in einem zweiten Schritt mit Triethylsilan/Trifluoressigsäure reduziert. Im finalen Schritt wurden zwei Schutzgruppen mit Bortrichlorid in DCM abgespaltet und [18F]VK5 als injektionsfertige Lösung in isotoner NaCl-Lösung erhalten. Es wurden radiochemische Ausbeuten von 0,7-1 % (EOS) nach einer durchschnittlichen Synthesedauer von 275 Minuten erhalten.rnDer Radioligand [18F]VK5 wurde anschließend in Autoradiographie-Versuchen an Hirnschnitten der Ratte hinsichtlich seiner Spezifität für die GABA-Bindungsstelle untersucht. Die unspezifische Bindung wurde durch die Zugabe von GABA bestimmt wonach kein signifikanter Unterschied festgestellt werden konnte. Die hohe unspezifische Bindung kann möglicherweise auf die niedrigen spezifischen Aktivitäten zurückgeführt werden. Diese lagen, bedingt durch die drei Schritte der Radiosynthese, in einem Bereich von 0,1-0,6 GBq/μmol. Die erhaltenen Ergebnisse lassen für zukünftige Versuche noch einige Optimierungsmöglichkeiten offen. Aufgrund der bisher erhaltenen Daten lässt sich daher keine definitive Aussage über die Eignung des Liganden [18F]VK5 als PET-Tracer treffen.rn

Problems in functioning from the patient perspective using the International Classification of Functioning, Disability and Health (ICF) as a reference

We conducted a qualitative, multicenter study using a focus group design to explore the lived experiences of persons with any kind of primary sleep disorder with regard to functioning and contextual factors using six open-ended questions related to the International Classification of Functioning, Disability and Health (ICF) components. We classified the results using the ICF as a frame of reference. We identified the meaningful concepts within the transcribed data and then linked them to ICF categories according to established linking rules. The six focus groups with 27 participants yielded a total of 6986 relevant concepts, which were linked to a total of 168 different second-level ICF categories. From the patient perspective, the ICF components: (1) Body Functions; (2) Activities & Participation; and (3) Environmental Factors were equally represented; while (4) Body Structures appeared poignantly less frequently. Out of the total number of concepts, 1843 concepts (26%) were assigned to the ICF component Personal Factors, which is not yet classified but could indicate important aspects of resource management and strategy development of those who have a sleep disorder. Therefore, treatment of patients with sleep disorders must not be limited to anatomical and (patho-)physiological changes, but should also consider a more comprehensive view that includes patient's demands, strategies and resources in daily life and the contextual circumstances surrounding the individual.

[Driving ability and daytime sleepiness]

Daytime sleepiness is a complaint of about 5-10% in a normal population. The consequences, such as impaired performance and accidents at the workplace and while driving, have major impact on the affected and on society. According to Swiss federal statistics only 1-3% of all motor vehicle accidents are due to excessive daytime sleepiness, which is in great contrast to a figure of 10 to 20% of all accidents derived from scientific studies. Due to the inadequate statistical representation of the problem, insufficient countermeasures have been realized, and the state of drivers breaching traffic regulations is not adequately investigated in this respect. The most prevalent cause of microsleep induced accidents is certainly lack of sleep due to social or professional reasons. A treating physician must also consider sedating drugs and various diseases. The typical characteristics of accidents due to falling asleep at the wheel and the risk factors involved are well established, so that informing the general public, taking prophylactic countermeasures and a targeted investigation in this respect of drivers who have breached the law are all feasible. Since symptoms of sleepiness can be recognized well before any impairment of performance occurs, the most important countermeasure is information of the drivers on the risk factors and on efficient countermeasures against sleepiness at the wheel. Besides correct diagnosis and treatment, the primary goal of physicians treating patients with pathological daytime sleepiness is to inform them at an early stage about the risks of sleepiness and the large responsibility they bear while driving. This information should be written down in the patients' records. Professional drivers suffering from daytime sleepiness, drivers who have already had an accident due to microsleep and unreasonable drivers should be referred to a centre of sleep disorders for objective measurements of sleepiness.

Sleepwalking in Parkinson's disease: a questionnaire-based survey

Sleepwalking (SW) corresponds to a complex sleep-associated behavior that includes locomotion, mental confusion, and amnesia. SW is present in about 10% of children and 2-3% of adults. In a retrospective series of 165 patients with Parkinson's disease (PD), we found adult-onset ("de novo") SW "de novo" in six (4%) of them. The aim of this study was to assess prospectively and systematically the frequency and characteristics of SW in PD patients. A questionnaire including items on sleep quality, sleep disorders, and specifically also SW and REM sleep behavior disorder (RBD), PD characteristics and severity, was sent to the members of the national PD patients organization in Switzerland. In the study, 36/417 patients (9%) reported SW, of which 22 (5%) had adult-onset SW. Patients with SW had significantly longer disease duration (p = 0.035), they reported more often hallucinations (p = 0.004) and nightmares (p = 0.003), and they had higher scores, suggestive for RBD in a validated questionnaire (p = 0.001). Patients with SW were also sleepier (trend to a higher Epworth Sleepiness Scale score, p = 0.055). Our data suggest that SW in PD patients is (1) more common than in the general population, and (2) is associated with RBD, nightmares, and hallucinations. Further studies including polysomnographic recordings are needed to confirm the results of this questionnaire-based analysis, to understand the relationship between SW and other nighttime wandering behaviors in PD, and to clarify the underlying mechanisms.

Clinical, polysomnographic and genome-wide association analyses of narcolepsy with cataplexy: a European Narcolepsy Network study

The aim of this study was to describe the clinical and PSG characteristics of narcolepsy with cataplexy and their genetic predisposition by using the retrospective patient database of the European Narcolepsy Network (EU-NN). We have analysed retrospective data of 1099 patients with narcolepsy diagnosed according to International Classification of Sleep Disorders-2. Demographic and clinical characteristics, polysomnography and multiple sleep latency test data, hypocretin-1 levels, and genome-wide genotypes were available. We found a significantly lower age at sleepiness onset (men versus women: 23.74 ± 12.43 versus 21.49 ± 11.83, P = 0.003) and longer diagnostic delay in women (men versus women: 13.82 ± 13.79 versus 15.62 ± 14.94, P = 0.044). The mean diagnostic delay was 14.63 ± 14.31 years, and longer delay was associated with higher body mass index. The best predictors of short diagnostic delay were young age at diagnosis, cataplexy as the first symptom and higher frequency of cataplexy attacks. The mean multiple sleep latency negatively correlated with Epworth Sleepiness Scale (ESS) and with the number of sleep-onset rapid eye movement periods (SOREMPs), but none of the polysomnographic variables was associated with subjective or objective measures of sleepiness. Variant rs2859998 in UBXN2B gene showed a strong association (P = 1.28E-07) with the age at onset of excessive daytime sleepiness, and rs12425451 near the transcription factor TEAD4 (P = 1.97E-07) with the age at onset of cataplexy. Altogether, our results indicate that the diagnostic delay remains extremely long, age and gender substantially affect symptoms, and that a genetic predisposition affects the age at onset of symptoms.

Urinary metabolites and antioxidant products of exogenous melatonin in the mouse

Exogenous melatonin is widely used for sleep disorders and has potential value in neuroprotection, cardioprotection and as an antioxidant. Here, a novel method is described for the determination of melatonin and six metabolites in mouse urine by use of LC-MS/MS and GC-MS. LC-MS/MS is used for the measurement of melatonin, N1-acetyl-5-methoxykynuramine (AMK), N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and 6-hydroxymelatonin (6-HMEL), while GC/MS is used for the determination of N-[2-(5-methoxy-2-oxo-2,3-dihydro-1H-indol-3-yl)-ethyl]-acetamide (2-OMEL) and cyclic 3-hydroxymelatonin (3-HMEL) with detection limits on column of 0.02-0.5 pmol, depending on the metabolite. Following oral administration of melatonin to mice, a 0-24 hr urine collection revealed the presence of melatonin (0.2% dose), 6-HMEL (37.1%) and NAS (3.1%) comprising >90% of the total metabolites; AMK and AFMK were also detected at 0.01% each; 2-OMEL was found at 2.2% of the dose, which is >100 times more than the AMK/AFMK pathway, and comprises >5% of the melatonin-related material detected in mouse urine. 3-HMEL was largely found as a sulfate conjugate. These studies establish sensitive assays for determination of six melatonin metabolites in mouse urine and confirm the potential for antioxidant activity of melatonin through the identification in vivo of AMK and AFMK, ring-opened metabolites with a high capacity for scavenging reactive oxygen species.

Influence of CYP2B6 polymorphism on plasma and intracellular concentrations and toxicity of efavirenz and nevirapine in HIV-infected patients

BACKGROUND: Efavirenz (EFV) and nevirapine (NVP) are metabolized by cytochrome P450 2B6 (CYP2B6). Allele 516 G>T (Gln172His) is associated with diminished activity of this isoenzyme, and may lead to differences in drug exposure. METHODS: We evaluated this allele as a pharmacogenetic marker of EFV and NVP pharmacokinetics and EFV toxicity in 167 participants receiving EFV and 59 receiving NVP recruited within the genetics project of the Swiss HIV Cohort Study. Drug concentrations were measured in plasma and in peripheral blood mononuclear cells (PBMCs) from the same sample. Neuropsychological toxicity of EFV (sleep disorders, mood disorders, fatigue) was assessed using a standardized questionnaire. RESULTS AND CONCLUSIONS: CYP2B6 516TT was associated with greater plasma and intracellular exposure to EFV, and greater plasma exposure to NVP. Intracellular drug concentration, and CYP2B6 genotype were predictors of EFV neuropsychological toxicity. CYP2B6 genotyping may be useful to complement an individualization strategy based on plasma drug determinations to increase the safety and tolerability of EFV.

The retina in Parkinson's disease

As a more complete picture of the clinical phenotype of Parkinson's disease emerges, non-motor symptoms have become increasingly studied. Prominent among these non-motor phenomena are mood disturbance, cognitive decline and dementia, sleep disorders, hyposmia and autonomic failure. In addition, visual symptoms are common, ranging from complaints of dry eyes and reading difficulties, through to perceptual disturbances (feelings of presence and passage) and complex visual hallucinations. Such visual symptoms are a considerable cause of morbidity in Parkinson's disease and, with respect to visual hallucinations, are an important predictor of cognitive decline as well as institutional care and mortality. Evidence exists of visual dysfunction at several levels of the visual pathway in Parkinson's disease. This includes psychophysical, electrophysiological and morphological evidence of disruption of retinal structure and function, in addition to disorders of ‘higher’ (cortical) visual processing. In this review, we will draw together work from animal and human studies in an attempt to provide an insight into how Parkinson's disease affects the retina and how these changes might contribute to the visual symptoms experienced by patients.

Mechanism-based inhibition of the melatonin rhythm enzyme: Pharmacologic exploitation of active site functional plasticity

Serotonin N-acetyltransferase is the enzyme responsible for the diurnal rhythm of melatonin production in the pineal gland of animals and humans. Inhibitors of this enzyme active in cell culture have not been reported previously. The compound N-bromoacetyltryptamine was shown to be a potent inhibitor of this enzyme in vitro and in a pineal cell culture assay (IC50 ≈ 500 nM). The mechanism of inhibition is suggested to involve a serotonin N-acetyltransferase-catalyzed alkylation reaction between N-bromoacetyltryptamine and reduced CoA, resulting in the production of a tight-binding bisubstrate analog inhibitor. This alkyltransferase activity is apparently catalyzed at a functionally distinct site compared with the acetyltransferase activity active site on serotonin N-acetyltransferase. Such active site plasticity is suggested to result from a subtle conformational alteration in the protein. This plasticity allows for an unusual form of mechanism-based inhibition with multiple turnovers, resulting in “molecular fratricide.” N-bromoacetyltryptamine should serve as a useful tool for dissecting the role of melatonin in circadian rhythm as well as a potential lead compound for therapeutic use in mood and sleep disorders.

Exceptionally potent inhibitors of fatty acid amide hydrolase: The enzyme responsible for degradation of endogenous oleamide and anandamide

The development of exceptionally potent inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of oleamide (an endogenous sleep-inducing lipid), and anandamide (an endogenous ligand for cannabinoid receptors) is detailed. The inhibitors may serve as useful tools to clarify the role of endogenous oleamide and anandamide and may prove to be useful therapeutic agents for the treatment of sleep disorders or pain. The combination of several features—an optimal C12–C8 chain length, π-unsaturation introduction at the corresponding arachidonoyl Δ8,9/Δ11,12 and oleoyl Δ9,10 location, and an α-keto N4 oxazolopyridine with incorporation of a second weakly basic nitrogen provided FAAH inhibitors with Kis that drop below 200 pM and are 102–103 times more potent than the corresponding trifluoromethyl ketones.

A doença de Parkinson no doente idoso

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Doença inflamatória intestinal pediátrica e comorbilidades psiquiátricas

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Snoring and its association with asthma in Indigenous children living in the Torres Strait and Northern Peninsula Area

Objective: Respiratory health of Indigenous and minority ethnic groups in affluent countries is poorer than their non-minority counterparts and sleep disorders are no exception. In children, obstructive sleep apnoea has the potential to result in serious long-term consequences. In 1999, we studied 1650 children and adolescents living in the Torres Strait and the Northern Peninsula Area, Australia. Here we report prevalence of snoring in these communities and relate its association with asthma symptoms. Methods: A population-based cross-sectional study was conducted in the Torres Strait region. Five indigenous communities were randomly selected and information was collected using a structured face-to-face interview based on a standardized questionnaire. There was a 98% response rate, and 1650 children, 0-17 years of age, were included in the study. Results: Overall, the prevalence of snoring was 14.2% (95% CI 12.5-15.9); 3.6% (95% CI 2.7-4.6) reported snorting, and 6% (95% CI 4.9-7.2) reported restless sleep. The prevalence of snoring was significantly higher among males (17.1% for males and 10.8 for females, P = 0.005). Children were five times more likely to have experienced snoring and snorting if they reported wheezing in the last 12 months. Conclusion: We conclude that the prevalence of symptoms suggestive of obstructive sleep problems is relatively high in children of this region. This highlights the need for awareness among the community patients and physicians about the problem of obstructive sleep-disordered breathing, especially in children with asthma, and for the need for further studies to measure prevalence of sleep breathing disorders among Indigenous Australians.

Genome-wide association analysis identifies novel loci for chronotype in 100,420 individuals from the UK Biobank

Our sleep timing preference, or chronotype, is a manifestation of our internal biological clock. Variation in chronotype has been linked to sleep disorders, cognitive and physical performance, and chronic disease. Here we perform a genome-wide association study of self-reported chronotype within the UK Biobank cohort (n=100,420). We identify 12 new genetic loci that implicate known components of the circadian clock machinery and point to previously unstudied genetic variants and candidate genes that might modulate core circadian rhythms or light-sensing pathways. Pathway analyses highlight central nervous and ocular systems and fear-response-related processes. Genetic correlation analysis suggests chronotype shares underlying genetic pathways with schizophrenia, educational attainment and possibly BMI. Further, Mendelian randomization suggests that evening chronotype relates to higher educational attainment. These results not only expand our knowledge of the circadian system in humans but also expose the influence of circadian characteristics over human health and life-history variables such as educational attainment.

Trabalho e saúde: análise da relação entre condições de trabalho e motivação no emprego e suas interfaces com a saúde dos profissionais nos hospitais públicos

Health results from the interaction of biological, social, economic, political and cultural factors. Under this perspective, we aim to analyze the relationship among working in public emergency hospitals and the health-sickness of the professionals who work there. We are based in a quantitative and qualitative research, in which 240 health professionals (doctors, nurses, social workers, psychologists, dentists, nutritionists, audiologists, physiotherapists and occupational therapists) answered a survey. All of them recognize the importance of work to guarantee favorable conditions to good health. However, they highlight its physical and mental wear effects on workers like stress, absence of a healthy life-style, high blood pressure, musculoskeletal, gastrointestinal and sleep disorders. It becomes urgent to face this reality, to enhance professionals' health and, consequently, the quality of user’s assistance, since the illness of health workers is strongly correlated with the existing health model in society.