The discovery of the ocular hypotensive effect of topically applied prostaglandins (PGs) has raised a number of questions about the mechanisms of action involved. The aim of the present thesis was to answer some of these questions. PGs reduce the intraocular pressure (IOP) by increasing uveoscleral flow through the ciliary muscle, but the exact mechanism is not known. Morphological changes may be involved. PGs are also involved in the inflammatory response. In the first study the aim was to investigate the effect of latanoprost, a prostaglandin F2 a-analogue, on the blood-aqueous barrier and the IOP restoration after long-term treatment. 26 glaucoma patients were treated with latanoprost (50 pg/ml) once daily for 6-12 months. Aqueous protein concentration was followed with a laser flare meter in 16 patients throughout this period. No change was observed. IOP increased slowly after withdrawal of treatment. It was concluded that latanoprost has no clinically significant effect on the permeability of the blood-aqueous barrier and that the IOP will return to pretreatment levels within a few weeks, indicating that any changes in the ciliary muscle morphology are reversible. In 20 healthy volunteers it was attempted to prevent the ocular hypotensive effect of latanoprost by inhibiting uveoscleral flow by a pronounced ciliary muscle contraction. For this purpose a high dose of the cholinergic agonist, physostigmine (1 drop 8 mg/ml alternate hours) was used. However, the effects on IOP of the two drugs were mainly additive most likely due to a short-lasting effect of physostigmine on the ciliary muscle. The progressive IOP reduction by physostigmine in the second study raised the question as to whether the drug reduces aqueous flow apart from enhancing outflow. On the contrary, in the third study repeated administrations of physostigmine, in 20 normal subjects, increased aqueous flow, measured with fluorophotometry, by about 25%. From studies of patients it is known that latanoprost twice daily has less ocular hypotensive effect than once daily. This was the subject of the two remaining studies. The possibility that latanoprost causes a short-lasting increase in aqueous flow was examined in 18 healthy volunteers. Application of a second drop in the morning would blunt some of the early IOP lowering effect of latanoprost. Once or twice daily applications had similar effect on aqueous flow, a tendency to an increase without any difference between the dose regimens. The next study confirmed the difference in effect on IOP between once and twice daily applications in 40 normal subjects. The difference remained even when one of the two applications was omitted after two weeks’ treatment. The results indicate that applying latanoprost twice daily induces a modest receptor desensitisation. / <p>Diss. Umeå : Umeå universitet, 1997, härtill 5 uppsatser.</p> / digitalisering@umu

Störungen der Aufwachphase sind sowohl in der Human-, als auch Veterinäranästhesiologie von Bedeutung. So kann eine verlängerte, aber auch eine unruhige exzitatorische Aufwachphase zusätzlichen intensivmedizinischen, zeitlichen und finanziellen Aufwand bedeuten. Eine Vielzahl von metabolischen und neuropathologischen, aber auch pharmakologischen Insulten werden insbesondere beim Menschen als Ursachen derartiger Aufwachstörungen beschrieben. Unter anderem scheint auch ein zentraler Acetylcholinmangel mitverantwortlich zu sein. Dieser Mangel wird durch die Anwendung von Physostigmin als indirektes, zentral wirkendes Parasympathomimetikum behoben. Inhalt dieser Studie ist der Einsatz von Physostigmin in der Aufwachphase des Hundes nach zwei gängigen l-Methadon-Narkoseprotokollen. Zum einen sollten Wirkungen und Nebenwirkungen des Medikamentes untersucht werden. Zum anderen sollte geklärt werden, ob Physostigmin in der Lage ist, die Aufwachphase in beiden Narkoseregimen zu verkürzen und auftretende Aufwachstörungen beseitigen zu können. Daraus resultierend war zu klären, ob ein Teil der vorhandenen Aufwachstörungen als Folge einer zentral anticholinergen Blockade auftreten und dem Zentral Anticholinergischen Syndrom (ZAS) zugeordnet werden können. Methodik: Die Untersuchung wurde an 40 klinisch gesunden Hunden (ASA I, II) nach elektiven Eingriffen oder Verfahren bildgebender Diagnostik in der postoperativen Aufwachphase durchgeführt. Zwanzig Tiere erhielten zur Narkoseeinleitung Diazepam und Levomethadon (DLA-Gruppe), zwanzig weitere Acepromazin und Levomethadon (ALA-Gruppe). Im Blindversuch wurde jeweils zehn Tieren pro Narkosegruppe Physostigmin (0,04 mg/kg KM als intravenöse Kurzinfusion über 10 Minuten, Gruppen DLAVerum, ALAVerum) und den anderen zehn Tieren 0,9%-ige Kochsalzlösung (Gruppen DLAPlacebo, ALAPlacebo) verabreicht. Die Untersuchungen beinhalteten die Messungen der Vitalparameter (Herzfrequenz, -rhythmus, arterieller systolischer und diastolischer Blutdruck, kapilläre Rückfüllzeit, Schleimhautfarbe Zusammenfassung 94 und innere Körpertemperatur), des Sedations- und Algesiegrades sowie Laboruntersuchungen (arterielle Blutgasanalytik, Säure-Base-Status, Hämatologie und klinische Chemie) und klinische Nebenwirkungen des Physostigmins. Darüber hinaus wurde das Aufwachverhalten beurteilt und mit dem ebenfalls dokumentierten Einschlafverhalten verglichen. Die Daten wurden in der ersten Stunde nach Applikation der Prüfsubstanz im Abstand von 10 Minuten sowie 120 und 180 Minuten nach Applikation erfasst. Hämatologische und klinisch chemische Untersuchungen erfolgten als Ausgangswerte vor Applikation sowie nach 60 Minuten. Ergebnisse: DLAVerum-Tiere besitzen unter Physostigmingabe einen signifikant niedrigeren Sedationsgrad im Messintervall 10 bis 40 Minuten (p< 0,05). Diese Tiere sind bereits 15 Minuten nach Beginn der Untersuchung in der Lage den Kopf zu heben (DLAPlacebo 30 Minuten). Im ALA-Regime konnten hier keine Unterschiede beobachtet werden. Der Algesiegrad aller vier Gruppen ist annähernd gleich. Nur zu drei Messzeiten ist dieser in DLAVerum um 1-2 Grade über der DLAPlacebo (0-27 Punkte). Das Aufwachverhalten unterscheidet sich nicht signifikant zwischen den Physostigmin-behandelten und -unbehandelten Tieren der beiden Narkoseregime und auch nicht zum Einschlafverhalten. In DLAVerum steigt die Herzfrequenz (nach initialem Abfall) wie auch der systolische Blutdruck tendenziell an (nicht signifikant). DLAPlacebo und ALAPlacebo zeigen konstante Herzfrequenzen und Blutdrücke. Signifikante Unterschiede fehlen zwischen DLAVerum und DLAPlacebo. ALAVerum hat signifikant niedrigere Blutdrücke zum Zeitpunkt 10 bis 40 Minuten als ALAPlacebo (p< 0,05). Schleimhautfarbe, Pulsqualität und Körpertemperatur unterscheiden sich nicht zwischen Physostigmin-behandelten und -unbehandelten Tieren beider Anästhesiegruppen (p> 0,05), verbessern sich jedoch mit Zunahme der Untersuchungszeit. Alle Tiere besitzen eine ausgeprägte respiratorische Azidose zu Untersuchungsbeginn. Signifikante Unterschiede bestehen zwischen DLAVerum und DLAPlacebo. Der paCO2 und der pH-Wert sind nach Physostigmingabe signifikant näher am Referenzbereich. Unterschiede zwischen Verum und Placebo fehlen im ALA-Regime. Bei der angewendeten Dosis wurden als Nebenwirkungen Brady- und Tachykardien beobachtet. Zusammenfassend kann ein antisedativer Effekt nur im Diazepam-l-Methadon-Regime unter Physostigmingabe beobachtet werden. Physostigmin lindert die atemdepressive Wirkung des Opioids Levomethadon in der DLAVerum-Gruppe. Derartige Effekte fehlen in der Acepromazin- l-Methadon-Narkose. In dieser Narkose wird einzig die blutdrucksenkende Wirkung des Acepromazins deutlich verstärkt, was bei alten oder kreislauf-insuffizienten Tieren von Bedeutung sein könnte. Unruhezustände, Lautäußerungen und Muskelzuckungen sind mit und ohne Physostigmin in gleichem Maße vorhanden. Ein zentraler Acetylcholinmangel kann für die beobachteten Aufwachstörungen nicht verantwortlich gemacht werden, da in diesem Fall Physostigmin eine Wirkung zeigen müsste. Ein „Zentral Anticholinergisches Syndrom“ lag somit nicht vor. / Disorders of anaesthetic recovery are found in human but also in veterinary medicine. It is important to understand, that a prolonged or an excitatory stage of recovery causes an additional time in post anaesthesia care unit and financial effort. Animals with agitation represent a risk factor for anaesthesia staff. Many metabolic, neuropathological and pharmacological insults cause these disorders in humans. A lack of acetylcholine within the brain seems to be a cause of arousal disorders. The therapy of choice is the application of physostigmine, as an indirect parasympathomimetic drug. The purpose of this study was the examination of the application of physostigmine in anaesthetic recovery of two l-methadone-anaesthetic regimes. Effects and side effects of physostigmine application in dogs should be described. The following questions should be answered additionally: Is physostigmine able to reduce the time of anaesthetic recovery? Can physostigmine restore disorders of anaesthetic recovery? Is the central anticholinergic syndrome (CAS) a cause of these disorders of anaesthetic recovery? Methods: The investigations were carried out on 40 clinically healthy dogs (ASA I & II) undergoing anaesthesia for elective surgery or diagnostic imaging. Patients were allocated to two different induction protocols: DLA (diazepam-l-methadone) and ALA (acepromacine-lmethadone). In both groups (n=20) 10 dogs received blinded either Physostigmine (n=10; verum) or isotonic saline (n=10; placebo) intravenously in randomized fashion. Following parameters were measured in postoperative period: heart rate, systolic and diastolic blood pressure and respiratory rate, colour of mucous membranes, capillary filling time and rectal body temperature. Analysis of arterial blood gas values and acid base status were performed. A modified numeric rating scale was used for calculating the sedation depth and a Summary 96 cumulative scale for measuring the intensity of algesia. Furthermore a comparison between sleep behaviour and arousal behaviour had been drawn. All parameters were evaluated at time 0 (beginning of application of verum versus placebo), every 10 minutes within the first hour and after the second and third hour. Blood samples (haematological, clinical chemistry) were collected at time point 0 and 60. Side effects of medication were documented. Results: There was a significant lower degree of sedation in DLAVerum compared to DLAPlacebo (p< 0.05), but not in ALA. The degree of pain in DLAVerum is two points over DLAPlacebo. No significant difference among the four groups has been found in behaviour of arousal, vocalization and the incidence of tremor and seizures. Heart rate and blood pressure increase none significantly in DLAVerum in contrast to DLAPlacebo (constant values). A significant reduction in systolic and diastolic blood pressure in ALAVerum, but not in ALAPlacebo was noticeable. Blood pressure in DLAVerum and DLAPlacebo was significant higher than in ALAVerum and ALAPlacebo. There was no significant difference between physostigmine treated and placebo treated animals in pulse quality, rectal body temperature and colour of mucous membranes. A respiratory acidosis has been found in nearly all dogs at the beginning of the investigation. Physostigmine has significantly reduced this opioid induced respiratory depression in DLA but not in ALA. The influence of Physostigmine in reversal of sedation, respiratory depression and restoration of blood pressure was time dependant. Differences were seen in time point 10 to 40 (50) minutes. Side effects were bradycardia and tachycardia, but not nausea or vomiting in contrast to human literature. Conclusion: The antisedative effect has been found only in diazepam-l-methadone-group, while the pressure depressant influence only in acepromacine-l-methadone group became obvious. Physostigmine intensifies the pressure depressant effect of acepromacine. Changes in blood pressure occurred in physical limits only but should be considered in cardiac and circulation insufficient patients. In this study it could be demonstrated that physostigmine could not resolve arousal disorders like restlessness, vocalization, tremor and seizures in dogs. A central lack of acetylcholine (a central anticholinergic syndrome) can not be therefore the cause of these problems. Physostigmine should be limited to severe agitation and prolonged times of arousal cases. Other causes of arousal disorders should be excluded before treatment. After application of physostigmine the supervision with ecg and blood pressure monitoring seems to be necessary.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2018-07-06T16:35:49Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) sueli_ferreira_fonseca.pdf: 2207045 bytes, checksum: cbbd1940c41e5fff7fb9ea96ff6381c6 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2018-07-18T13:14:36Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) sueli_ferreira_fonseca.pdf: 2207045 bytes, checksum: cbbd1940c41e5fff7fb9ea96ff6381c6 (MD5) / Made available in DSpace on 2018-07-18T13:14:36Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) sueli_ferreira_fonseca.pdf: 2207045 bytes, checksum: cbbd1940c41e5fff7fb9ea96ff6381c6 (MD5) Previous issue date: 2017 / Existem evid?ncias que a estimula??o colin?rgica central aumenta a dissipa??o de calor em ratos normotensos como consequ?ncia de altera??es cardiovasculares via modula??o da atividade barorreflexa. No entanto, n?o h? dados publicados sobre o envolvimento do sistema colin?rgico central nestas respostas em modelo experimental que apresenta altera??o da sensibilidade dos barorreceptores e d?ficit termorregulat?rio. Assim, o objetivo do presente estudo foi verificar o envolvimento do sistema colin?rgico central na modula??o das repostas cardiovasculares e termorregulat?rias durante o repouso e exerc?cio f?sico em ratos espontaneamente hipertensos (SHR). Ratos Wistar machos (n = 33) e SHR (n = 33) foram implantados com uma c?nula intracerebroventricular (icv) para inje??es de 2 ?L de fisostigmina (fis) ou solu??o salina (sal). Temperaturas da cauda (Tcauda) e corporal interna (Tint), press?o arterial sist?lica (PAS), frequ?ncia card?aca (FC) e taxa metab?lica foram registradas durante os 60 minutos em que os ratos permaneceram em repouso, bem como durante o exerc?cio f?sico at? a fadiga ap?s inje??es icv randomizadas. Na situa??o repouso, o tratamento com fis iniciou uma sucess?o de respostas cardiovasculares e termorregulat?rias que resultaram em aumento da PAS, redu??o da FC e aumento de Tcauda nos grupos Wistar e SHR. A magnitude da ativa??o desses mecanismos foi mais intensa no SHR, afetando a Tint e melhorando a dissipa??o de calor. Durante o exerc?cio f?sico, o tratamento com fis foi capaz de modular as repostas cardiovasculares promovendo aumento significativo da PAS, seguido de bradicardia reflexa em ratos SHR e Wistar. Estas respostas foram mais intensas nos ratos Wistar. N?o houve diferen?a significativa para a Tcauda e Tint no grupo SHR fis em rela??o ao grupo sal. Entretanto, fis impactou positivamente no desempenho f?sico. Em conjunto, esses resultados fornecem evid?ncias que, durante a situa??o de repouso, a estimula??o colin?rgica central modula as repostas termorregulat?rias por meio de mudan?as no sistema cardiovascular de ratos Wistar e SHR, sendo que essas respostas s?o mais acentuadas em ratos SHR impactando na dissipa??o de calor. Durante o exerc?cio f?sico, a administra??o central de fis promove altera??es no sistema cardiovascular de ratos normotensos e hipertensos. Apesar dessas altera??es n?o terem sido suficientes para ajustar as respostas termorregulat?rias em ratos SHR, impactaram positivamente no desempenho f?sico. / Tese (Doutorado) ? Programa Multic?ntrico de P?s-Gradua??o em Ci?ncias Fisiol?gicas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / There is evidence that central cholinergic stimulation increases heat dissipation in normotensive rats through changes on the cardiovascular system via modulation of baroreceptors function. However, there is no published data regarding the involvement of the central cholinergic system in cardiovascular and thermoregulatory responses in spontaneously hypertensive rats (SHR), which are animals that possess altered baroreceptor sensitivity and thermoregulatory deficit. Therefore, the aim of this study was to verify the involvement of the central cholinergic system in cardiovascular and thermoregulatory adjustments in SHR. Male Wistar (n = 33) and SHR (n = 33) rats were implanted with an intracerebroventricular (icv) cannula for injections of 2 ?L of physostigmine (phy) or saline (sal) solution. Tail (Ttail) and internal body temperatures (Tint), systolic arterial pressure (SAP), heart rate (HR) and metabolic rate were registered during 60 minutes while the animals remained at rest and during exercise until fatigue after randomly receiving the injections. Phy treatment started a succession of cardiovascular and thermoregulatory responses that resulted in increased SAP, reduced HR and increased Ttail in both Wistar and SHR groups. The magnitude of the activation of these mechanisms seems to be more intense in SHR, even affecting the Tint, and improve heat dissipation. During physical exercise, the phy treatment was able to modulate the cardiovascular responses promoting a significant increase of SAP, followed by reflex bradycardia in SHR and Wistar rats. These responses were more intense in Wistar rats. There was no significant difference for Ttail and Tint in the SHR group, however, phy positively impacted the physical performance. Taken together, these results provide evidence that at rest the central cholinergic stimulation modulates thermoregulatory responses through changes in the cardiovascular system of Wistar and SHR rats, and SHR rats presented greater cardiovascular and thermoregulatory responses than normotensive rats after central cholinergic stimulation. During physical exercise the central administration of phy promotes adjustments in the cardiovascular system of normotensive and hypertensive rats. Although these adjustments were not sufficient to pair the thermoregulatory responses in SHR rats, they had a positive impact on physical performance.

The effect of 1,2,3,4-tetrahydro-9-aminoacridine (THA) on quantal transmitter release was examined at the frog neuromuscular junction. THA (3 μM) caused an increase in m (no. of quanta released) as measured by K+-evoked miniature endplate potential (MEPP) frequency. This was due to an increase in p (probability of release), as n (no. of functional release sites) was unchanged. The increase in p was dose-dependent over a range of 0.3-10 μM. By contrast, physostigmine (3 μM) caused a decrease in p, and neostigmine, which does not cross the nerve membrane, had no consistent effect on p. At the postsynaptic site, neostigmine produced the largest increase in MEPP size (79.2%), and THA produced the smallest (17.5%). The divergent effects of THA and physostigmine on p indicate a fundamental difference in their actions at the nerve terminal.

Störungen der Aufwachphase sind sowohl in der Human-, als auch Veterinäranästhesiologie von Bedeutung. So kann eine verlängerte, aber auch eine unruhige exzitatorische Aufwachphase zusätzlichen intensivmedizinischen, zeitlichen und finanziellen Aufwand bedeuten. Eine Vielzahl von metabolischen und neuropathologischen, aber auch pharmakologischen Insulten werden insbesondere beim Menschen als Ursachen derartiger Aufwachstörungen beschrieben. Unter anderem scheint auch ein zentraler Acetylcholinmangel mitverantwortlich zu sein. Dieser Mangel wird durch die Anwendung von Physostigmin als indirektes, zentral wirkendes Parasympathomimetikum behoben. Inhalt dieser Studie ist der Einsatz von Physostigmin in der Aufwachphase des Hundes nach zwei gängigen l-Methadon-Narkoseprotokollen. Zum einen sollten Wirkungen und Nebenwirkungen des Medikamentes untersucht werden. Zum anderen sollte geklärt werden, ob Physostigmin in der Lage ist, die Aufwachphase in beiden Narkoseregimen zu verkürzen und auftretende Aufwachstörungen beseitigen zu können. Daraus resultierend war zu klären, ob ein Teil der vorhandenen Aufwachstörungen als Folge einer zentral anticholinergen Blockade auftreten und dem Zentral Anticholinergischen Syndrom (ZAS) zugeordnet werden können. Methodik: Die Untersuchung wurde an 40 klinisch gesunden Hunden (ASA I, II) nach elektiven Eingriffen oder Verfahren bildgebender Diagnostik in der postoperativen Aufwachphase durchgeführt. Zwanzig Tiere erhielten zur Narkoseeinleitung Diazepam und Levomethadon (DLA-Gruppe), zwanzig weitere Acepromazin und Levomethadon (ALA-Gruppe). Im Blindversuch wurde jeweils zehn Tieren pro Narkosegruppe Physostigmin (0,04 mg/kg KM als intravenöse Kurzinfusion über 10 Minuten, Gruppen DLAVerum, ALAVerum) und den anderen zehn Tieren 0,9%-ige Kochsalzlösung (Gruppen DLAPlacebo, ALAPlacebo) verabreicht. Die Untersuchungen beinhalteten die Messungen der Vitalparameter (Herzfrequenz, -rhythmus, arterieller systolischer und diastolischer Blutdruck, kapilläre Rückfüllzeit, Schleimhautfarbe Zusammenfassung 94 und innere Körpertemperatur), des Sedations- und Algesiegrades sowie Laboruntersuchungen (arterielle Blutgasanalytik, Säure-Base-Status, Hämatologie und klinische Chemie) und klinische Nebenwirkungen des Physostigmins. Darüber hinaus wurde das Aufwachverhalten beurteilt und mit dem ebenfalls dokumentierten Einschlafverhalten verglichen. Die Daten wurden in der ersten Stunde nach Applikation der Prüfsubstanz im Abstand von 10 Minuten sowie 120 und 180 Minuten nach Applikation erfasst. Hämatologische und klinisch chemische Untersuchungen erfolgten als Ausgangswerte vor Applikation sowie nach 60 Minuten. Ergebnisse: DLAVerum-Tiere besitzen unter Physostigmingabe einen signifikant niedrigeren Sedationsgrad im Messintervall 10 bis 40 Minuten (p< 0,05). Diese Tiere sind bereits 15 Minuten nach Beginn der Untersuchung in der Lage den Kopf zu heben (DLAPlacebo 30 Minuten). Im ALA-Regime konnten hier keine Unterschiede beobachtet werden. Der Algesiegrad aller vier Gruppen ist annähernd gleich. Nur zu drei Messzeiten ist dieser in DLAVerum um 1-2 Grade über der DLAPlacebo (0-27 Punkte). Das Aufwachverhalten unterscheidet sich nicht signifikant zwischen den Physostigmin-behandelten und -unbehandelten Tieren der beiden Narkoseregime und auch nicht zum Einschlafverhalten. In DLAVerum steigt die Herzfrequenz (nach initialem Abfall) wie auch der systolische Blutdruck tendenziell an (nicht signifikant). DLAPlacebo und ALAPlacebo zeigen konstante Herzfrequenzen und Blutdrücke. Signifikante Unterschiede fehlen zwischen DLAVerum und DLAPlacebo. ALAVerum hat signifikant niedrigere Blutdrücke zum Zeitpunkt 10 bis 40 Minuten als ALAPlacebo (p< 0,05). Schleimhautfarbe, Pulsqualität und Körpertemperatur unterscheiden sich nicht zwischen Physostigmin-behandelten und -unbehandelten Tieren beider Anästhesiegruppen (p> 0,05), verbessern sich jedoch mit Zunahme der Untersuchungszeit. Alle Tiere besitzen eine ausgeprägte respiratorische Azidose zu Untersuchungsbeginn. Signifikante Unterschiede bestehen zwischen DLAVerum und DLAPlacebo. Der paCO2 und der pH-Wert sind nach Physostigmingabe signifikant näher am Referenzbereich. Unterschiede zwischen Verum und Placebo fehlen im ALA-Regime. Bei der angewendeten Dosis wurden als Nebenwirkungen Brady- und Tachykardien beobachtet. Zusammenfassend kann ein antisedativer Effekt nur im Diazepam-l-Methadon-Regime unter Physostigmingabe beobachtet werden. Physostigmin lindert die atemdepressive Wirkung des Opioids Levomethadon in der DLAVerum-Gruppe. Derartige Effekte fehlen in der Acepromazin- l-Methadon-Narkose. In dieser Narkose wird einzig die blutdrucksenkende Wirkung des Acepromazins deutlich verstärkt, was bei alten oder kreislauf-insuffizienten Tieren von Bedeutung sein könnte. Unruhezustände, Lautäußerungen und Muskelzuckungen sind mit und ohne Physostigmin in gleichem Maße vorhanden. Ein zentraler Acetylcholinmangel kann für die beobachteten Aufwachstörungen nicht verantwortlich gemacht werden, da in diesem Fall Physostigmin eine Wirkung zeigen müsste. Ein „Zentral Anticholinergisches Syndrom“ lag somit nicht vor.:Inhaltsverzeichnis I Inhaltsverzeichnis 1 EINLEITUNG UND FRAGESTELLUNG 1 2 LITERATURÜBERSICHT 4 2.1 Physostigmin 4 2.1.1 Geschichte 4 2.1.2 Chemische Struktur und Charakteristik 5 2.1.3 Anwendungsgebiete 5 2.1.4 Wirkungen und Pharmakodynamik 6 2.1.5 Pharmakokinetik 8 2.1.6 Neben- und Wechselwirkungen 9 2.1.7 Kontraindikation 11 2.2 Das Zentral Anticholinergische Syndrom 12 2.2.1 Definition 12 2.2.2 Symptome 12 2.2.3 Vorkommen 13 2.2.4 Ursachen 13 2.2.5 Differentialdiagnosen 14 2.2.6 Therapie 15 2.2.7 Prophylaxe und Prognose 15 2.2.8 Das Zentral Anticholinergische Syndrom beim Hund 15 2.3 Die Aufwachphase der Anästhesie 16 2.3.1 Die physiologische Aufwachphase 16 2.3.2 Die abnormale Aufwachphase 17 2.3.2.1 Krämpfe und Exzitationen 17 2.3.2.2 Fehlende Spontanatmung und Atemstillstand 18 2.3.2.3 Langanhaltender Nachschlaf und postanästhetische Bewusstseinsstörungen 18 2.3.2.4 Das vorschnelle Erwachen 19 2.3.2.5 Störungen der Thermoregulation 19 2.3.2.6 Metabolische Störungen und Organdysfunktionen 19 2.3.3 Beurteilung der Aufwachphase 20 Inhaltsverzeichnis II 2.4 Wirkung ausgewählter Anästhetika 22 2.4.1 Diazepam 22 2.4.2 Acepromazin 22 2.4.3 Levomethadon 23 3 EIGENE UNTERSUCHUNGEN 24 3.1 Patienten, Material und Methodik 24 3.1.1 Patienten 24 3.1.2 Untersuchungsgruppen 26 3.1.3 Randomisierung 27 3.1.4 Untersuchungsablauf 27 3.1.5 Untersuchte Parameter und Messmethoden 29 3.1.5.1 Herz-Kreislauf-Funktionen 29 3.1.5.2 Atmungsapparat 30 3.1.5.3 Schleimhautfarbe und kapilläre Rückfüllungszeit 30 3.1.5.4 Innere Körpertemperatur 30 3.1.5.5 Sedationsgrad 30 3.1.5.6 Schmerzgrad 32 3.1.5.7 Einschlaf- und Aufwachverhalten 34 3.1.5.8 Blutuntersuchungen 34 3.1.6 Nebenwirkungen 36 3.1.7 Statistische Auswertung 36 4 ERGEBNISSE 38 4.1 Biographische Daten 38 4.1.1 Alter 38 4.1.2 Geschlecht 38 4.1.3 Körpermasse 38 4.1.4 Rasse 39 4.1.5 Art der Untersuchung und Operation 39 4.1.6 Anästhesie- und Operationszeit 39 4.1.7 Lokal- und Regionalanästhesien, Schmerzmittelapplikation 40 4.1.8 Einsatz von Anticholinergika 41 4.1.9 Einsatz von Inhalationsanästhetika und Injektionsanästhetika 41 Inhaltsverzeichnis III 4.2 Vitalparameter 41 4.2.1 Herzfrequenz, -rhythmus und Pulsqualität 41 4.2.2 Systolischer und diastolischer Blutdruck 44 4.2.3 Atemfrequenz 47 4.2.4 Schleimhautfarbe und kapilläre Rückfüllungszeit 49 4.2.5 Innere Körpertemperatur 49 4.3 Sedationsgrad 51 4.4 Schmerzgrad 54 4.5 Vergleich des Einschlaf- und Aufwachverhaltens 56 4.6 Laboruntersuchungen 57 4.6.1 Analyse der Blutgase und des Säure-Basen-Status 57 4.6.1.1 pH-Wert 57 4.6.1.2 Arterieller Kohlendioxid-Partialdruck 59 4.6.1.3 Arterieller Sauerstoff-Partialdruck 62 4.6.1.4 Arterielle Sauerstoffsättigung 63 4.6.1.5 Standardbikarbonatkonzentration 63 4.6.1.6 Aktueller Basenüberschuss 64 4.6.2 Hämatologische Untersuchung 64 4.6.3 Klinisch-chemische Untersuchungen 67 4.7 Nebenwirkungen 71 5 DISKUSSION 73 5.1 Methodik 73 5.1.1 Untersuchte Tiere 73 5.1.2 Untersuchte Parameter und Messmethoden 74 5.1.2.1 Vitalparameter 74 5.1.2.2 Laboruntersuchungen 766 5.1.2.3 Sedations- und Analgesiescore, Einschlaf- und Aufwachverhalten 77 5.1.3 Untersuchungsgruppen, Anästhesieprotokolle und Physostigmin-Dosierung 78 5.1.4 Datenerfassung, Statistik und graphischen Darstellung 79 Inhaltsverzeichnis IV 5.2 Diskussion der Ergebnisse 79 5.2.1 Sedation 79 5.2.2 Analgesie 81 5.2.3 Aufwachverhalten 822 5.2.4 Vitalparameter 83 5.2.4.1 Herz-Kreislauffunktionen 83 5.2.4.2 Atmungsapparat 85 5.2.4.3 Schleimhautfarbe 86 5.2.4.4 Innere Körpertemperatur 86 5.2.5 Laboruntersuchungen 86 5.2.6 Nebenwirkungen 90 5.3 Klinische Schlussfolgerungen 91 5.4 Praxisrelevanz 92 6 ZUSAMMENFASSUNG 93 7 SUMMARY 95 8 LITERATURVERZEICHNIS 98 9 ANHANG 119 9.1 Verzeichnis der Tabellen 119 9.2 Tabellen 122 9.3 Verzeichnis der Abbildungen 167 9.4 Anhang der Abbildungen / Disorders of anaesthetic recovery are found in human but also in veterinary medicine. It is important to understand, that a prolonged or an excitatory stage of recovery causes an additional time in post anaesthesia care unit and financial effort. Animals with agitation represent a risk factor for anaesthesia staff. Many metabolic, neuropathological and pharmacological insults cause these disorders in humans. A lack of acetylcholine within the brain seems to be a cause of arousal disorders. The therapy of choice is the application of physostigmine, as an indirect parasympathomimetic drug. The purpose of this study was the examination of the application of physostigmine in anaesthetic recovery of two l-methadone-anaesthetic regimes. Effects and side effects of physostigmine application in dogs should be described. The following questions should be answered additionally: Is physostigmine able to reduce the time of anaesthetic recovery? Can physostigmine restore disorders of anaesthetic recovery? Is the central anticholinergic syndrome (CAS) a cause of these disorders of anaesthetic recovery? Methods: The investigations were carried out on 40 clinically healthy dogs (ASA I & II) undergoing anaesthesia for elective surgery or diagnostic imaging. Patients were allocated to two different induction protocols: DLA (diazepam-l-methadone) and ALA (acepromacine-lmethadone). In both groups (n=20) 10 dogs received blinded either Physostigmine (n=10; verum) or isotonic saline (n=10; placebo) intravenously in randomized fashion. Following parameters were measured in postoperative period: heart rate, systolic and diastolic blood pressure and respiratory rate, colour of mucous membranes, capillary filling time and rectal body temperature. Analysis of arterial blood gas values and acid base status were performed. A modified numeric rating scale was used for calculating the sedation depth and a Summary 96 cumulative scale for measuring the intensity of algesia. Furthermore a comparison between sleep behaviour and arousal behaviour had been drawn. All parameters were evaluated at time 0 (beginning of application of verum versus placebo), every 10 minutes within the first hour and after the second and third hour. Blood samples (haematological, clinical chemistry) were collected at time point 0 and 60. Side effects of medication were documented. Results: There was a significant lower degree of sedation in DLAVerum compared to DLAPlacebo (p< 0.05), but not in ALA. The degree of pain in DLAVerum is two points over DLAPlacebo. No significant difference among the four groups has been found in behaviour of arousal, vocalization and the incidence of tremor and seizures. Heart rate and blood pressure increase none significantly in DLAVerum in contrast to DLAPlacebo (constant values). A significant reduction in systolic and diastolic blood pressure in ALAVerum, but not in ALAPlacebo was noticeable. Blood pressure in DLAVerum and DLAPlacebo was significant higher than in ALAVerum and ALAPlacebo. There was no significant difference between physostigmine treated and placebo treated animals in pulse quality, rectal body temperature and colour of mucous membranes. A respiratory acidosis has been found in nearly all dogs at the beginning of the investigation. Physostigmine has significantly reduced this opioid induced respiratory depression in DLA but not in ALA. The influence of Physostigmine in reversal of sedation, respiratory depression and restoration of blood pressure was time dependant. Differences were seen in time point 10 to 40 (50) minutes. Side effects were bradycardia and tachycardia, but not nausea or vomiting in contrast to human literature. Conclusion: The antisedative effect has been found only in diazepam-l-methadone-group, while the pressure depressant influence only in acepromacine-l-methadone group became obvious. Physostigmine intensifies the pressure depressant effect of acepromacine. Changes in blood pressure occurred in physical limits only but should be considered in cardiac and circulation insufficient patients. In this study it could be demonstrated that physostigmine could not resolve arousal disorders like restlessness, vocalization, tremor and seizures in dogs. A central lack of acetylcholine (a central anticholinergic syndrome) can not be therefore the cause of these problems. Physostigmine should be limited to severe agitation and prolonged times of arousal cases. Other causes of arousal disorders should be excluded before treatment. After application of physostigmine the supervision with ecg and blood pressure monitoring seems to be necessary.:Inhaltsverzeichnis I Inhaltsverzeichnis 1 EINLEITUNG UND FRAGESTELLUNG 1 2 LITERATURÜBERSICHT 4 2.1 Physostigmin 4 2.1.1 Geschichte 4 2.1.2 Chemische Struktur und Charakteristik 5 2.1.3 Anwendungsgebiete 5 2.1.4 Wirkungen und Pharmakodynamik 6 2.1.5 Pharmakokinetik 8 2.1.6 Neben- und Wechselwirkungen 9 2.1.7 Kontraindikation 11 2.2 Das Zentral Anticholinergische Syndrom 12 2.2.1 Definition 12 2.2.2 Symptome 12 2.2.3 Vorkommen 13 2.2.4 Ursachen 13 2.2.5 Differentialdiagnosen 14 2.2.6 Therapie 15 2.2.7 Prophylaxe und Prognose 15 2.2.8 Das Zentral Anticholinergische Syndrom beim Hund 15 2.3 Die Aufwachphase der Anästhesie 16 2.3.1 Die physiologische Aufwachphase 16 2.3.2 Die abnormale Aufwachphase 17 2.3.2.1 Krämpfe und Exzitationen 17 2.3.2.2 Fehlende Spontanatmung und Atemstillstand 18 2.3.2.3 Langanhaltender Nachschlaf und postanästhetische Bewusstseinsstörungen 18 2.3.2.4 Das vorschnelle Erwachen 19 2.3.2.5 Störungen der Thermoregulation 19 2.3.2.6 Metabolische Störungen und Organdysfunktionen 19 2.3.3 Beurteilung der Aufwachphase 20 Inhaltsverzeichnis II 2.4 Wirkung ausgewählter Anästhetika 22 2.4.1 Diazepam 22 2.4.2 Acepromazin 22 2.4.3 Levomethadon 23 3 EIGENE UNTERSUCHUNGEN 24 3.1 Patienten, Material und Methodik 24 3.1.1 Patienten 24 3.1.2 Untersuchungsgruppen 26 3.1.3 Randomisierung 27 3.1.4 Untersuchungsablauf 27 3.1.5 Untersuchte Parameter und Messmethoden 29 3.1.5.1 Herz-Kreislauf-Funktionen 29 3.1.5.2 Atmungsapparat 30 3.1.5.3 Schleimhautfarbe und kapilläre Rückfüllungszeit 30 3.1.5.4 Innere Körpertemperatur 30 3.1.5.5 Sedationsgrad 30 3.1.5.6 Schmerzgrad 32 3.1.5.7 Einschlaf- und Aufwachverhalten 34 3.1.5.8 Blutuntersuchungen 34 3.1.6 Nebenwirkungen 36 3.1.7 Statistische Auswertung 36 4 ERGEBNISSE 38 4.1 Biographische Daten 38 4.1.1 Alter 38 4.1.2 Geschlecht 38 4.1.3 Körpermasse 38 4.1.4 Rasse 39 4.1.5 Art der Untersuchung und Operation 39 4.1.6 Anästhesie- und Operationszeit 39 4.1.7 Lokal- und Regionalanästhesien, Schmerzmittelapplikation 40 4.1.8 Einsatz von Anticholinergika 41 4.1.9 Einsatz von Inhalationsanästhetika und Injektionsanästhetika 41 Inhaltsverzeichnis III 4.2 Vitalparameter 41 4.2.1 Herzfrequenz, -rhythmus und Pulsqualität 41 4.2.2 Systolischer und diastolischer Blutdruck 44 4.2.3 Atemfrequenz 47 4.2.4 Schleimhautfarbe und kapilläre Rückfüllungszeit 49 4.2.5 Innere Körpertemperatur 49 4.3 Sedationsgrad 51 4.4 Schmerzgrad 54 4.5 Vergleich des Einschlaf- und Aufwachverhaltens 56 4.6 Laboruntersuchungen 57 4.6.1 Analyse der Blutgase und des Säure-Basen-Status 57 4.6.1.1 pH-Wert 57 4.6.1.2 Arterieller Kohlendioxid-Partialdruck 59 4.6.1.3 Arterieller Sauerstoff-Partialdruck 62 4.6.1.4 Arterielle Sauerstoffsättigung 63 4.6.1.5 Standardbikarbonatkonzentration 63 4.6.1.6 Aktueller Basenüberschuss 64 4.6.2 Hämatologische Untersuchung 64 4.6.3 Klinisch-chemische Untersuchungen 67 4.7 Nebenwirkungen 71 5 DISKUSSION 73 5.1 Methodik 73 5.1.1 Untersuchte Tiere 73 5.1.2 Untersuchte Parameter und Messmethoden 74 5.1.2.1 Vitalparameter 74 5.1.2.2 Laboruntersuchungen 766 5.1.2.3 Sedations- und Analgesiescore, Einschlaf- und Aufwachverhalten 77 5.1.3 Untersuchungsgruppen, Anästhesieprotokolle und Physostigmin-Dosierung 78 5.1.4 Datenerfassung, Statistik und graphischen Darstellung 79 Inhaltsverzeichnis IV 5.2 Diskussion der Ergebnisse 79 5.2.1 Sedation 79 5.2.2 Analgesie 81 5.2.3 Aufwachverhalten 822 5.2.4 Vitalparameter 83 5.2.4.1 Herz-Kreislauffunktionen 83 5.2.4.2 Atmungsapparat 85 5.2.4.3 Schleimhautfarbe 86 5.2.4.4 Innere Körpertemperatur 86 5.2.5 Laboruntersuchungen 86 5.2.6 Nebenwirkungen 90 5.3 Klinische Schlussfolgerungen 91 5.4 Praxisrelevanz 92 6 ZUSAMMENFASSUNG 93 7 SUMMARY 95 8 LITERATURVERZEICHNIS 98 9 ANHANG 119 9.1 Verzeichnis der Tabellen 119 9.2 Tabellen 122 9.3 Verzeichnis der Abbildungen 167 9.4 Anhang der Abbildungen

Na literatura encontram-se referências de estudos envolvendo micro-organismos endofíticos, e mais recentemente estudos que avaliam a interação entre micro-organismos o que resulta na modificação, no tipo ou quantidade dos compostos que são produzidos. Neste trabalho foram realizados cultivos simples e mistos do fungo Colletotrichum boninense FLe 8.1 e da actinobacteria Streptomyces albospinus RLe 7, endófiticos isolados de Lychnophora ericoides que pertencem à coleção do Laboratório de Química de Microorganismos (LQMo) da FCFRP-USP, com o objetivo de aumentar suas capacidades de produção de novos compostos com atividade biológica. O cultivo misto, ou co-cultivo, é uma estratégia que tem sido usada para o acesso aos produtos naturais de origem microbiana. Existem poucos relatos de compostos com atividade biológica isolados a partir de S. albospinus e não há relatos de metabólitos secundários obtidos a partir de C. boninense. Nenhum desses micro-organismos tem sido descrito como endofítico na literatura e não existem relatos sobre co-cultivos envolvendo qualquer um deles na busca de compostos bioativos. Juntando as informações geradas através das diferentes técnicas de detecção utilizadas, como TLC, HPLC-DAD, GC-MS, ESI-MS, RMN e depois da análise correspondente, foi possível identificar e atribuir as estruturas de algumas substâncias conhecidas de origem microbiana como a mevalonolactona, o tirosol, a fisostigmina, a desferrioxamina E. ESI-MS foi utilizada para análises dos extratos brutos originados dos cultivos em meio arroz parbolizado permitindo visualizar compostos produzidos em baixas quantidades pelos micro-organismos na cultura simples quanto na co-cultura. Além disso, foram obtidos os perfis metabólicos desses micro-organismos a partir de cultivos em placa de Petri, possibilitando a detecção dos metabólitos em regiões específicas da interação microbiana, o que conduziu à identificação da fisostigmina e o seu análogo N-etilcarbamato produzidos pela actinobactéria, na interação com o fungo. Análises posteriores de MS sequencial permitiram obter perfis de fragmentação, os quais, juntamente com os dados dos íons detectados nos extratos, foram comparados com a informação disponível em bases de dados como DNP, METLIN e MassBank. Tais informações geradas a partir dessas análises permitiram sugerir possíveis compostos envolvidos na interação dos micro-organismos endofíticos mencionados. Foi sugerido que a fisostigmina, substância produzida pela actinobactéria S. albospinus RLe 7 e isolada nesse trabalho, poderia ter algum papel na inibição do crescimento do fungo C. boninense FLe 8.1 já que a inibição só foi observada quando cultivados ambos os micro-organismos na mesma placa de Petri. Porém, bioensaios com fisostigmina pura demonstraram que essa substância não possui atividade antifúngica per se, mas é possível que exista uma sinergia com outras substancias produzidas pela actinobactéria. Os resultados apresentados nesse trabalho demonstram a importância do uso de técnicas de detecção muito sensíveis, como a ESI-MS, na identificação de substâncias envolvidas na troca metabólica e permitiram gerar informações que serão utilizadas em estudos futuros utilizando esses micro-organismos endofíticos. / There are several studies involving endophytic microorganisms, and more recently some studies evaluate microbial interaction resulting in metabolic profiles modifications. In this study, simple and mixed cultures of the fungus Colletotrichum boninense FLe 8.1 and the actinobacteria Streptomyces albospinus RLe 7 were carried out. These endophytic microorganisms, isolated from Lychnophora ericoides, belong to the collection of the Laboratory of Microbial Chemistry (LQMo) of the FCFRP-USP. The main goal of this work was to increase the bioactive compounds production capacities. Co-culture, or mixed culture, is a recent strategy for accessing microbial natural products. There are few reports of bioactive metabolites from S. albospinus RLe 7 and there are no one about secondary metabolites obtained from C. boninense FLe 8.1. Neither the actinobacteria nor the fungus have been described as endophytic microorganisms and there are no co-culture studies involving these microorganisms in order to obtain natural products. Joining and analyzing all together the generated information from several detection techniques, such as TLC, HPLCDAD, GC-MS, ESI-MS, NMR, it was possible to identify and attribute the chemical structures of some known natural, such as mevalonolactone, tyrosol, physostigmine and deferrioxamine E. ESI-MS was used for extracts analysis from cultures in parboiled rice medium, allowing visualization of compounds produced in very low yields. Besides that, it was possible to obtain metabolic profiles from cultures in Petri dishes, leading to the detection of metabolites in specific regions of the microbial interaction. Physostigmine and its N-ethylcarbamate analogue were identified as actinobacteria metabolites in the interaction against the fungus. Posterior analysis by MS/MS allowed to obtain fragmentation profiles, which together with the ions detected from the extracts analysis, were compared by searching in databases, such as DNP, METLIN and MassBank. Such information allowed to suggest possible candidates for the compounds involved in the microbial metabolic exchange. It was hypothesized that physostigmine, isolated from the actinobacteria in this work, had a role in the fungal growth inhibition observed when both microorganisms were co-cultured. However, this substance did not show considerable antifungal activity when tested against C. boninense FLe 8.1, but it is possible that other compounds produced by this actinobacteria are acting in a synergistic way. The results showed here demonstrated the importance of very sensitive techniques, as ESI-MS, in the identification of molecules involved in the microbial metabolic exchange and will help in the generation of information for future studies involving those endophytic microorganisms.

Na literatura encontram-se referências de estudos envolvendo micro-organismos endofíticos, e mais recentemente estudos que avaliam a interação entre micro-organismos o que resulta na modificação, no tipo ou quantidade dos compostos que são produzidos. Neste trabalho foram realizados cultivos simples e mistos do fungo Colletotrichum boninense FLe 8.1 e da actinobacteria Streptomyces albospinus RLe 7, endófiticos isolados de Lychnophora ericoides que pertencem à coleção do Laboratório de Química de Microorganismos (LQMo) da FCFRP-USP, com o objetivo de aumentar suas capacidades de produção de novos compostos com atividade biológica. O cultivo misto, ou co-cultivo, é uma estratégia que tem sido usada para o acesso aos produtos naturais de origem microbiana. Existem poucos relatos de compostos com atividade biológica isolados a partir de S. albospinus e não há relatos de metabólitos secundários obtidos a partir de C. boninense. Nenhum desses micro-organismos tem sido descrito como endofítico na literatura e não existem relatos sobre co-cultivos envolvendo qualquer um deles na busca de compostos bioativos. Juntando as informações geradas através das diferentes técnicas de detecção utilizadas, como TLC, HPLC-DAD, GC-MS, ESI-MS, RMN e depois da análise correspondente, foi possível identificar e atribuir as estruturas de algumas substâncias conhecidas de origem microbiana como a mevalonolactona, o tirosol, a fisostigmina, a desferrioxamina E. ESI-MS foi utilizada para análises dos extratos brutos originados dos cultivos em meio arroz parbolizado permitindo visualizar compostos produzidos em baixas quantidades pelos micro-organismos na cultura simples quanto na co-cultura. Além disso, foram obtidos os perfis metabólicos desses micro-organismos a partir de cultivos em placa de Petri, possibilitando a detecção dos metabólitos em regiões específicas da interação microbiana, o que conduziu à identificação da fisostigmina e o seu análogo N-etilcarbamato produzidos pela actinobactéria, na interação com o fungo. Análises posteriores de MS sequencial permitiram obter perfis de fragmentação, os quais, juntamente com os dados dos íons detectados nos extratos, foram comparados com a informação disponível em bases de dados como DNP, METLIN e MassBank. Tais informações geradas a partir dessas análises permitiram sugerir possíveis compostos envolvidos na interação dos micro-organismos endofíticos mencionados. Foi sugerido que a fisostigmina, substância produzida pela actinobactéria S. albospinus RLe 7 e isolada nesse trabalho, poderia ter algum papel na inibição do crescimento do fungo C. boninense FLe 8.1 já que a inibição só foi observada quando cultivados ambos os micro-organismos na mesma placa de Petri. Porém, bioensaios com fisostigmina pura demonstraram que essa substância não possui atividade antifúngica per se, mas é possível que exista uma sinergia com outras substancias produzidas pela actinobactéria. Os resultados apresentados nesse trabalho demonstram a importância do uso de técnicas de detecção muito sensíveis, como a ESI-MS, na identificação de substâncias envolvidas na troca metabólica e permitiram gerar informações que serão utilizadas em estudos futuros utilizando esses micro-organismos endofíticos. / There are several studies involving endophytic microorganisms, and more recently some studies evaluate microbial interaction resulting in metabolic profiles modifications. In this study, simple and mixed cultures of the fungus Colletotrichum boninense FLe 8.1 and the actinobacteria Streptomyces albospinus RLe 7 were carried out. These endophytic microorganisms, isolated from Lychnophora ericoides, belong to the collection of the Laboratory of Microbial Chemistry (LQMo) of the FCFRP-USP. The main goal of this work was to increase the bioactive compounds production capacities. Co-culture, or mixed culture, is a recent strategy for accessing microbial natural products. There are few reports of bioactive metabolites from S. albospinus RLe 7 and there are no one about secondary metabolites obtained from C. boninense FLe 8.1. Neither the actinobacteria nor the fungus have been described as endophytic microorganisms and there are no co-culture studies involving these microorganisms in order to obtain natural products. Joining and analyzing all together the generated information from several detection techniques, such as TLC, HPLCDAD, GC-MS, ESI-MS, NMR, it was possible to identify and attribute the chemical structures of some known natural, such as mevalonolactone, tyrosol, physostigmine and deferrioxamine E. ESI-MS was used for extracts analysis from cultures in parboiled rice medium, allowing visualization of compounds produced in very low yields. Besides that, it was possible to obtain metabolic profiles from cultures in Petri dishes, leading to the detection of metabolites in specific regions of the microbial interaction. Physostigmine and its N-ethylcarbamate analogue were identified as actinobacteria metabolites in the interaction against the fungus. Posterior analysis by MS/MS allowed to obtain fragmentation profiles, which together with the ions detected from the extracts analysis, were compared by searching in databases, such as DNP, METLIN and MassBank. Such information allowed to suggest possible candidates for the compounds involved in the microbial metabolic exchange. It was hypothesized that physostigmine, isolated from the actinobacteria in this work, had a role in the fungal growth inhibition observed when both microorganisms were co-cultured. However, this substance did not show considerable antifungal activity when tested against C. boninense FLe 8.1, but it is possible that other compounds produced by this actinobacteria are acting in a synergistic way. The results showed here demonstrated the importance of very sensitive techniques, as ESI-MS, in the identification of molecules involved in the microbial metabolic exchange and will help in the generation of information for future studies involving those endophytic microorganisms.

Alzheimer’s disease (AD) is a chronic, progressive neurodegenerative disorder, characterized clinically by a progressive loss of memory, cognitive function, ability to care for oneself and psychiatric symptoms. First-line agents for the treatment of AD are ChE inhibitors (DON, GAL), whose modest clinical efficacy and the high incidence of dose-limiting toxicities limit their clinical utility. In addition to AD, ChE inhibitors (PYR) are used for other medical conditions, such as myasthenia gravis (MG). Furthermore, ChE inhibitors (PYR) are used by military personnel prophylactically if impending exposure to chemical warfare agents, e.g., soman, is suspected. The purpose of this research project was to understand the effect of age on the in-vitro sensitivity of ChE inhibitors in human RBCs and plasma. Understanding possible covariates, such as age and gender, may assist in optimizing dosing regimens of ChE inhibitors and/or developing newer ChE inhibitors with better adverse effect profiles. Plasma PHYS concentrations were measured by a validated HPLC-FD method. RBC AChE activity and plasma BuChE activity were measured by a modified Ellman’s colorimetric method using the model substrates, acetylthiocholine and butyrylthiocholine, respectively. The kinetics of RBC and plasma ChE activity followed Michaelis-Menten kinetics. Acetylthiocholine was found to be a nonselective substrate (RBC AChE Km = 73 μM; plasma BuChE Km = 117 μM); while butyrylthiocholine was a selective substrate for plasma BuChE (RBC AChE Km = 130,000 μM; plasma BuChE Km = 72 μM). For the following studies, RBC AChE activity was measured using acetylthiocholine as the substrate and plasma BuChE activity was measured using butyrylthiocholine as the substrate. This research project was performed in two parts: First, mechanistic studies of PHYS, PYR, DON and GAL, explored and determined the mechanism of in-vitro inhibition of RBC AChE and plasma BuChE inhibition, as well as the in-vitro degradation of PHYS in human whole blood, plasma and RBC. PHYS was rapidly degraded in human whole blood, RBC and plasma and followed Michaelis-Menten kinetics but its degradation clearance - scaled to whole blood clearance - was only predicted to account for 4-6% (i.e., 195-261 ml/min) of the reported total body clearance for PHYS (4500 ml/min). RBCs were responsible for 60% of the whole blood clearance while plasma accounted for 40% of the whole blood clearance. Inhibition results indicated that both PHYS and PYR were nonselective and rapid suicide ChE inactivators. PYR inactivated RBC AChE more rapidly at low concentrations and inactivated plasma BuChE more rapidly at high concentrations, but inactivated both more rapidly than PHYS. PHYS was a more potent inactivator than PYR with a Ki for RBC AChE of 0.011 μM and 0.063 μM, respectively, and 0.023 μM and 0.036 μM, respectively for plasma BuChE. DON was found to be a noncompetitive inhibitor for RBC AChE (Ki,noncomp = 114 μM), but a competitive inhibitor for plasma BuChE (Ki,comp = 213 μM). GAL was found to be a competitive inhibitor for both RBC AChE (Ki,comp = 66 μM) and plasma BuChE (Ki,comp = 358 μM). The second part involved a clinical study with ten young and nine elderly healthy subjects, balanced for gender, who donated blood for an in-vitro study in order to assess any age- and gender-related differences in in-vitro sensitivity to RBC AChE and plasma BuChE inhibition to all four ChE inhibitors. Elderly adults were found to be 2-3-fold less sensitive compared to the young adults for PHYS (BuChE Ki,pss; 0.010 and 0.015 μM, young and elderly, respectively) and PYR (AChE Ki,pss; 0.12 and 0.25 μM, young and elderly, respectively) only, while neither DON nor GAL showed any age-related differences in sensitivity. The observed differences for PHYS and PYR may be due to kinetic differences in ChE inactivation between young and aged adults, rather then a difference in binding affinities/potencies. These carbamate ChE inhibitors, presumably, have a slower decarbamoylation rate in younger adults than elderly adults, which leads to the observed difference in in-vitro sensitivity. The above in-vitro results were consistent with results of a meta-analysis: In a study by Knapp et al. (1991), young males (n=6), receiving 18 mg, 24 mg and 30 mg PHYS tablets, showed similar ex-vivo plasma BuChE sensitivity to (28 %/(ng/ml)) as the in-vitro sensitivity for young males in the current study (33 %/(ng/ml)). On the other hand, in the study by Men (2004), elderly males (n=8) and females (n=8), receiving 6.7 μg/kg PHYS as 30-minute infusion, showed similar ex-vivo RBC AChE sensitivity (12 %/(ng/ml)) as the in-vitro sensitivity for elderly subjects in the current study (9.7 %/(ng/ml)). This suggests that in-vitro measurement of ChE sensitivity is predictive of ex-vivo sensitivity in clinical studies. The study results suggest that elderly adults may require a 2-3-fold higher blood concentration than young adults to achieve the same ChE inhibition. This may explain why for epistigmine, an investigational carbamate ChE inhibitor for the treatment of AD, the maximum tolerated dose observed in young adults (40 mg single dose) was lower than for older adults (90 mg/day). Higher sensitivity in young adults prevented further dose escalation, while all elderly subjects tolerated higher doses. This research may have implications for other diseases and conditions, most notably MG and as a prophylaxis of nerve gases poisoning. As patients with MG age, they may become less sensitive to PYR, the most common symptomatic treatment for MG, and an increase in dose may be required. Further, older military personnel assigned to receive PYR, may require increased doses to achieve the targeted 10% RBC AChE inhibition, necessary to protect against nerve gas poisoning.