Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Control Student: Pavla ábelová Supervisor: Assoc. Prof. RNDr. Veronika Opletalová, Ph.D. Supervisor specialist: PharmDr. Jan Korábe ný, Ph.D. Title of diploma thesis: Synthesis, biological evaluation and in silico studies in the series of novel 7-methoxytacrine-donepezile like compounds Alzheimer's disease (AD) is an irreversible neurodegenerative disorder of the brain characterized clinically by loss of memory, deterioration of activities of daily living and cognition. The pathological hallmarks of AD include neuritic plaques composed of extracellularly stored fibrils of amyloid- peptide, intracellular deposits of hyperphosphorylated tau and neurotransmitter deficits. The aim of the study was to design and synthesize 7-methoxytacrine-donepezil-like compounds as potential inhibitors of acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8). New compounds consist of 7-methoxytacrine representing less toxic derivative of tacrine and benzylpiperazine moiety corresponding to donepezil fragment. To determine the potential of new derivatives, AChE and BChE inhibitory activities of the new molecules were assessed in vitro according to the method of Ellman et al....

Donepezil is a centrally acting acetylcholinesterase (AChE) inhibitor with therapeutic potential in inflammatory diseases; however, the underlying autonomic and cholinergic mechanisms remain unclear. Here, we assessed effects of donepezil on mean arterial pressure (MAP), heart rate (HR), HR variability, and body temperature in conscious adult male C57BL/6 mice to investigate the autonomic pathways involved. Central versus peripheral cholinergic effects of donepezil were assessed using pharmacological approaches including comparison with the peripherally acting AChE inhibitor, neostigmine. Drug treatments included donepezil (2.5 or 5 mg/kg sc), neostigmine methyl sulfate (80 or 240 μg/kg ip), atropine sulfate (5 mg/kg ip), atropine methyl bromide (5 mg/ kg ip), or saline. Donepezil, at 2.5 and 5 mg/kg, decreased HR by 36 ± 4% and 44 ± 3% compared with saline (n = 10, P < 0.001). Donepezil, at 2.5 and 5 mg/kg, decreased temperature by 13 ± 2% and 22 ± 2% compared with saline (n = 6, P < 0.001). Modest (P < 0.001) increases in MAP were observed with donepezil after peak bradycardia occurred. Atropine sulfate and atropine methyl bromide blocked bradycardic responses to donepezil, but only atropine sulfate attenuated hypothermia. The pressor response to donepezil was similar in mice coadministered atropine sulfate; however, coadministration of atropine methyl bromide potentiated the increase in MAP. Neostigmine did not alter HR or temperature, but did result in early increases in MAP. Despite the marked bradycardia, donepezil did not increase normalized high-frequency HR variability. We conclude that donepezil causes marked bradycardia and hypothermia in conscious mice via the activation of muscarinic receptors while concurrently increasing MAP via autonomic and cholinergic pathways that remain to be elucidated.

Alzheimer’s disease is a neurodegenerative disorder characterized by symptoms such as memory loss and behavioral change, and it is the sixth leading cause of death in the U.S. This literature-based thesis aims to detail the history of the disease as well as pertinent information, such as basic brain histology, disease pathogenesis, and genetic profiles of victims. The paper will then discuss the available treatment options, from their annual costs and mechanisms of action to an evaluation of their cost-effectiveness. The information in this paper was collected through an online investigation of sources including research studies and medical journals. The treatments discussed in this thesis consist of six drugs: aducanumab, donepezil, rivastigmine, galantamine, memantine, and memantine-donepezil combination. Aducanumab is the only disease-modifying drug to receive FDA approval, but its efficacy is marred in controversy and it lacks cost-effectiveness. The remaining five drugs all have similar cost-effective values, but generic donepezil is an outlier with significantly better results. This paper’s findings indicate that generic donepezil is the most optimal treatment, but that further research should be conducted on aducanumab. Research also suggests that public health advocates must be vigorous in their attempts to make these drugs more affordable to the general population.

A deprivação maternal é conhecida por promover alterações neuroquímicas, comportamentais e da estrutura cerebral que persistem ao longo da vida. Aqui investigamos se os déficits cognitivos em ratos deprivados podem ser relacionados com uma disfunção no sistema colinérgico e na síntese de proteínas relacionadas aos processos mnemônicos. Ratas Wistar prenhas foram colocadas em caixas individuais e mantidas no biotério no ciclo claro/escuro (12:12 horas) com comida e água a vontade. Após o parto, as mães foram separadas dos seus filhotes por 3 horas, por dia, a partir do dia 1 pós-parto (PND-1) até o PND-10. Para fazer isto, as mães foram removidas das caixas residência e colocadas em uma caixa diferente, enquanto os filhotes permaneceram na caixa original, a qual foi transferida para outra sala mantida a temperatura de 32°C. Quando adultos com 120-150 d ias de idade, os animais machos deprivados e não deprivados foram divididos em duas etapas experimentais: 1) Foram sacrificados para medirmos a acetilcolinesterase (AchE) ou treinados no reconhecimento de objetos e no reconhecimento social, ou 2) Foram treinados e testados na esquiva inibitória e no Morris water maze ou foram treinados e sacrificados para mensurarmos o fator de transcrição CREB e a síntese das proteínas ERK1/2 após o treinamento em 3 diferentes tempos. Na primeira etapa, a deprivação maternal repetida afeta a memória no reconhecimento de objetos e no reconhecimento social. Além disso, ratos deprivados mostraram aumento na atividade da AchE no hipocampo e no córtex perirrinal. A administração oral de inibidores da AchE donepezil ou galantamina (1mg/kg) 30 min antes da sessão de treino reverteu os prejuízos na memória causados pela deprivação maternal. Na segunda etapa, mostramos que a deprivação maternal repetida também afeta a memória aversiva e espacial. Os animais treinados na esquiva inibitória mostraram déficits cognitivos quando testados na memória de curta (2 horas) e de longa duração (24 horas) após o treino. Além disso, a análise densitométrica das proteínas revelou que ratos deprivados treinados na esquiva não aumentaram a fosforilação de CREB e ERK1/2 após o treino. Da mesma forma, ratos deprivados não foram hábeis em reter o aprendizado reverso no labirinto aquático de Morris, mas aqui, os déficits cognitivos podem ser relacionados com o menor aumento da fosforilação do CREB e da ERK1/2 somente 2 horas após o treino no aprendizado reverso. Esses dados sugerem que a deprivação materna afeta os processos mnemônicos em ratos adultos, e que estes prejuízos podem ser mediados por modificações no sistema colinérgico ou na síntese de proteínas. / Maternal deprivation is known to result in long-lasting neurochemical, behavioral and brain structural effects. Here we investigated whether the cognitive aspects of these deficits might be related to a disruption of the cholinergic system and protein synthesis correlated with mnemonic process. Pregnant Wistar rats were individually housed and maintained on a 12:12 h light/dark cycle with food and water freely available. The mothers were separated from their pups for 3 h per day from postnatal day 1 (PND-1) to PND- 10. To do that, the dams were moved to a different cage and the pups maintained in the original home cage, which was transferred to a different room kept at 32ºC. After they reached 120-150 days of age, maternal-deprived and non-deprived male rats were either sacrificed for brain acetylcholinesterase (AchE) and protein (CREB and ERK1/2) measurement, or trained in an object recognition, social recognition, inhibitory avoidance and Morris water maze tasks and divided in two sets of experiments: 1) Complete repeated maternal deprivation affects recognition and social memory and there was increased acetylcholinesterase activity in hippocampus and perirhinal cortex of the deprived rats. Oral administration of the acetylcholinesterase inhibitors, donepezil or galantamine (1mg/kg) 30 min before training session reversed the memory impairments caused by maternal deprivation. 2) Complete maternal deprivation also affects aversive and spatial memory. These animals showed memory deficits in STM and LTM in inhibitory avoidance. Furthermore, densitometric analysis of proteins revealed that deprived rats did not increase the phosphorylation of ERK and CREB after training. Similarly, deprived rats did not able to retain the reversed learning in the Morris water maze but here, the cognitive deficit can be related with the increase in the protein phosphorylation measurement only 2 hours after training in reversed learning compared to non deprived rats. That findings suggest that maternal deprivation affects memory processing at adulthood and that this impairment can be mediated by modification of the cholinergic system or in the protein synthesis.

A deprivação maternal é conhecida por promover alterações neuroquímicas, comportamentais e da estrutura cerebral que persistem ao longo da vida. Aqui investigamos se os déficits cognitivos em ratos deprivados podem ser relacionados com uma disfunção no sistema colinérgico e na síntese de proteínas relacionadas aos processos mnemônicos. Ratas Wistar prenhas foram colocadas em caixas individuais e mantidas no biotério no ciclo claro/escuro (12:12 horas) com comida e água a vontade. Após o parto, as mães foram separadas dos seus filhotes por 3 horas, por dia, a partir do dia 1 pós-parto (PND-1) até o PND-10. Para fazer isto, as mães foram removidas das caixas residência e colocadas em uma caixa diferente, enquanto os filhotes permaneceram na caixa original, a qual foi transferida para outra sala mantida a temperatura de 32°C. Quando adultos com 120-150 d ias de idade, os animais machos deprivados e não deprivados foram divididos em duas etapas experimentais: 1) Foram sacrificados para medirmos a acetilcolinesterase (AchE) ou treinados no reconhecimento de objetos e no reconhecimento social, ou 2) Foram treinados e testados na esquiva inibitória e no Morris water maze ou foram treinados e sacrificados para mensurarmos o fator de transcrição CREB e a síntese das proteínas ERK1/2 após o treinamento em 3 diferentes tempos. Na primeira etapa, a deprivação maternal repetida afeta a memória no reconhecimento de objetos e no reconhecimento social. Além disso, ratos deprivados mostraram aumento na atividade da AchE no hipocampo e no córtex perirrinal. A administração oral de inibidores da AchE donepezil ou galantamina (1mg/kg) 30 min antes da sessão de treino reverteu os prejuízos na memória causados pela deprivação maternal. Na segunda etapa, mostramos que a deprivação maternal repetida também afeta a memória aversiva e espacial. Os animais treinados na esquiva inibitória mostraram déficits cognitivos quando testados na memória de curta (2 horas) e de longa duração (24 horas) após o treino. Além disso, a análise densitométrica das proteínas revelou que ratos deprivados treinados na esquiva não aumentaram a fosforilação de CREB e ERK1/2 após o treino. Da mesma forma, ratos deprivados não foram hábeis em reter o aprendizado reverso no labirinto aquático de Morris, mas aqui, os déficits cognitivos podem ser relacionados com o menor aumento da fosforilação do CREB e da ERK1/2 somente 2 horas após o treino no aprendizado reverso. Esses dados sugerem que a deprivação materna afeta os processos mnemônicos em ratos adultos, e que estes prejuízos podem ser mediados por modificações no sistema colinérgico ou na síntese de proteínas. / Maternal deprivation is known to result in long-lasting neurochemical, behavioral and brain structural effects. Here we investigated whether the cognitive aspects of these deficits might be related to a disruption of the cholinergic system and protein synthesis correlated with mnemonic process. Pregnant Wistar rats were individually housed and maintained on a 12:12 h light/dark cycle with food and water freely available. The mothers were separated from their pups for 3 h per day from postnatal day 1 (PND-1) to PND- 10. To do that, the dams were moved to a different cage and the pups maintained in the original home cage, which was transferred to a different room kept at 32ºC. After they reached 120-150 days of age, maternal-deprived and non-deprived male rats were either sacrificed for brain acetylcholinesterase (AchE) and protein (CREB and ERK1/2) measurement, or trained in an object recognition, social recognition, inhibitory avoidance and Morris water maze tasks and divided in two sets of experiments: 1) Complete repeated maternal deprivation affects recognition and social memory and there was increased acetylcholinesterase activity in hippocampus and perirhinal cortex of the deprived rats. Oral administration of the acetylcholinesterase inhibitors, donepezil or galantamine (1mg/kg) 30 min before training session reversed the memory impairments caused by maternal deprivation. 2) Complete maternal deprivation also affects aversive and spatial memory. These animals showed memory deficits in STM and LTM in inhibitory avoidance. Furthermore, densitometric analysis of proteins revealed that deprived rats did not increase the phosphorylation of ERK and CREB after training. Similarly, deprived rats did not able to retain the reversed learning in the Morris water maze but here, the cognitive deficit can be related with the increase in the protein phosphorylation measurement only 2 hours after training in reversed learning compared to non deprived rats. That findings suggest that maternal deprivation affects memory processing at adulthood and that this impairment can be mediated by modification of the cholinergic system or in the protein synthesis.

A deprivação maternal é conhecida por promover alterações neuroquímicas, comportamentais e da estrutura cerebral que persistem ao longo da vida. Aqui investigamos se os déficits cognitivos em ratos deprivados podem ser relacionados com uma disfunção no sistema colinérgico e na síntese de proteínas relacionadas aos processos mnemônicos. Ratas Wistar prenhas foram colocadas em caixas individuais e mantidas no biotério no ciclo claro/escuro (12:12 horas) com comida e água a vontade. Após o parto, as mães foram separadas dos seus filhotes por 3 horas, por dia, a partir do dia 1 pós-parto (PND-1) até o PND-10. Para fazer isto, as mães foram removidas das caixas residência e colocadas em uma caixa diferente, enquanto os filhotes permaneceram na caixa original, a qual foi transferida para outra sala mantida a temperatura de 32°C. Quando adultos com 120-150 d ias de idade, os animais machos deprivados e não deprivados foram divididos em duas etapas experimentais: 1) Foram sacrificados para medirmos a acetilcolinesterase (AchE) ou treinados no reconhecimento de objetos e no reconhecimento social, ou 2) Foram treinados e testados na esquiva inibitória e no Morris water maze ou foram treinados e sacrificados para mensurarmos o fator de transcrição CREB e a síntese das proteínas ERK1/2 após o treinamento em 3 diferentes tempos. Na primeira etapa, a deprivação maternal repetida afeta a memória no reconhecimento de objetos e no reconhecimento social. Além disso, ratos deprivados mostraram aumento na atividade da AchE no hipocampo e no córtex perirrinal. A administração oral de inibidores da AchE donepezil ou galantamina (1mg/kg) 30 min antes da sessão de treino reverteu os prejuízos na memória causados pela deprivação maternal. Na segunda etapa, mostramos que a deprivação maternal repetida também afeta a memória aversiva e espacial. Os animais treinados na esquiva inibitória mostraram déficits cognitivos quando testados na memória de curta (2 horas) e de longa duração (24 horas) após o treino. Além disso, a análise densitométrica das proteínas revelou que ratos deprivados treinados na esquiva não aumentaram a fosforilação de CREB e ERK1/2 após o treino. Da mesma forma, ratos deprivados não foram hábeis em reter o aprendizado reverso no labirinto aquático de Morris, mas aqui, os déficits cognitivos podem ser relacionados com o menor aumento da fosforilação do CREB e da ERK1/2 somente 2 horas após o treino no aprendizado reverso. Esses dados sugerem que a deprivação materna afeta os processos mnemônicos em ratos adultos, e que estes prejuízos podem ser mediados por modificações no sistema colinérgico ou na síntese de proteínas. / Maternal deprivation is known to result in long-lasting neurochemical, behavioral and brain structural effects. Here we investigated whether the cognitive aspects of these deficits might be related to a disruption of the cholinergic system and protein synthesis correlated with mnemonic process. Pregnant Wistar rats were individually housed and maintained on a 12:12 h light/dark cycle with food and water freely available. The mothers were separated from their pups for 3 h per day from postnatal day 1 (PND-1) to PND- 10. To do that, the dams were moved to a different cage and the pups maintained in the original home cage, which was transferred to a different room kept at 32ºC. After they reached 120-150 days of age, maternal-deprived and non-deprived male rats were either sacrificed for brain acetylcholinesterase (AchE) and protein (CREB and ERK1/2) measurement, or trained in an object recognition, social recognition, inhibitory avoidance and Morris water maze tasks and divided in two sets of experiments: 1) Complete repeated maternal deprivation affects recognition and social memory and there was increased acetylcholinesterase activity in hippocampus and perirhinal cortex of the deprived rats. Oral administration of the acetylcholinesterase inhibitors, donepezil or galantamine (1mg/kg) 30 min before training session reversed the memory impairments caused by maternal deprivation. 2) Complete maternal deprivation also affects aversive and spatial memory. These animals showed memory deficits in STM and LTM in inhibitory avoidance. Furthermore, densitometric analysis of proteins revealed that deprived rats did not increase the phosphorylation of ERK and CREB after training. Similarly, deprived rats did not able to retain the reversed learning in the Morris water maze but here, the cognitive deficit can be related with the increase in the protein phosphorylation measurement only 2 hours after training in reversed learning compared to non deprived rats. That findings suggest that maternal deprivation affects memory processing at adulthood and that this impairment can be mediated by modification of the cholinergic system or in the protein synthesis.

No description available.

Alzheimer´s disease (AD) is today the world’s most common disease within dementia as it affects most number of people that are demented. There are no cure to AD, just symptomatic treatment are available. Scientists are still discussing the reason behind AD and many different theories exist. The most popular one is the amyloid cascade hypothesis. To diagnose AD many different types of scales are used to determine a person´s cognitive skills, also blood- and spinal cord samples are taken. One of the most popular scale that is used to diagnose AD is named mini mental state exam (MMSE). MMSE consists of several domains including: orientation, reading, mental arithmetic, writing and figure copying. The maximum of points are 30 and a score of 24 or less indicates some type of dementia. Today acetylcholinesteraseinhibitors like donepezil, rivastigmine and galantamine are used to treat people with mild to moderate AD. People with severe AD are recommended treatment with a NMDA-receptor-antagonist, memantine. The result from this study includes a summary of results from five studies. All of them studied the effect of donepezil over time and three of them also studied donepezils effect compared to placebo. Patients that were included had mild- moderate AD and in one study they measured a continued treatment with donepezil. All studies measured the results with scales and tests, including MMSE, that assessed patient’s cognitive ability. The results showed that patients treated with donepezil had significantly improved their scores on the scale MMSE at the end of the studies compared from baseline in three studies. When compared with placebo patients treated with donepezil got significantly better scores on MMSE. These changes were small even if significantly proven. The conclusion of this work was that MMSE is not optimal for measuring changes in AD if the patient is treated with donepezil because of the small changes. MMSE have difficulties to detect the small changes (a change of ≤ 3 points) and therefore other scales and tests are in need. Donepezil showed a significantly improvement in the beginning of the studies but after a few weeks that improvement had decreased or disappeared. It could be in everyone´s favor to start medication in an early stage of the disease and then after a while make a new assessment of the patient’s health.

Inledning: Alzheimers sjukdomen (AD) är den vanligaste demenssjukdomen och beskrevs först år 1906 av Alois Alzheimer. AD är en neurodegenerativ sjukdom orsakad av obalans mellan produktion och utsöndring av proteinet beta-amyloid. Lagrat beta-amyloid bildar senila plack i hjärncellerna. Fosforylerat tau bildar neurofibrillära nystan i nervcellers cellkroppar. Koncentrationen av beta-amyloid, fosforylerat tau och tau mäts i cerebrospinalvätskan och används för att kunna följa sjukdomsförloppet vid demenssjukdomar. Vid AD finns en brist på signalsubstansen acetylkolin i hjärnan och en minskning av substanser som serotonin, noradrenalin och dopamin. Det är ämnen som behövs för att nervceller ska kunna kommunicera. Funktionen av glutamaterg neurotransmission är påverkad, vilket inducerar symtom och sjukdomsprogression. Det vanligaste symtomet är minnesproblem, det förekommer även koncentrationssvårigheter och uppmärksamhetsstörningar i stressade situationer. Sjukdomsförloppet är indelat i tre olika stadier, mild, måttlig och svår. Vid misstänkt AD-diagnos görs en demensutredning inom primärvården. Det görs en basal och en utvidgad demensutredning. Det är vanligt att använda sig av olika skattningsskalor för att ställa diagnos, men även för att övervaka sjukdomsförloppet samt utvärdera behandlingseffekt. De olika skattningsskalorna som finns mäter kognition, beteendesymtom och funktionsförändring. Det finns flera riskfaktorer för att drabbas av AD och ökad ålder är den främsta. Syfte: Syftet var att utvärdera effekt och säkerhet av donepezil i monoterapi vid behandling av måttlig till svår alzheimers sjukdom. Metod: Arbetet baserades på litteratursökning i databasen PubMed. Sökord som användes var ”Alzheimer’s disease”, ”severe”, ”donepezil” och ”monotherapy”. Det var 5 artiklar som inkluderades. Resultat: På Severe Impairment Battery som användes i fyra av fem studier gav donepezil en förbättring jämfört med placebo. Förändringen var statistiskt signifikant i samtliga studier. Mini mental state examination användes i studie 1 – 3 och 5 där donepezil gav en förbättring i studie 1 – 3 men en försämring i studie 5. I studie 5 var försämringen mindre än den för placebo. Samtliga studier visade statistiskt signifikanta resultat. På Alzheimer´s disease cooperative study – activities of daily living som användes i studie 2 – 4 gav donepezil en försämring. Det var bara studie 3 som visade statistiskt signifikanta resultat. Neuropsychiatric Inventory användes i studie 1 – 3 och donepezil gav förbättring i samtliga studier. I studie 2 var förbättringen mindre än den för placebo, men i studie 1 och 3 gav donepezil en större förbättring än placebo. Det var bara studie 1 som visade statistiskt signifikanta resultat. Samtliga studier i det här arbetet rapporterade biverkningar och det var liknande för patienterna som behandlats med donepezil och för dem som behandlats med placebo. De flesta biverkningarna som rapporterades var milda till måttliga. Studie 5 rapporterade flest allvarliga biverkningar men ingen av dem ansågs vara kopplade till behandlingen av donepezil. Donepezil anses vara ett säkert läkemedel vid behandling av måttlig till svår AD. Effekten är liten vid svår AD men donepezil ger en långsammare försämring jämfört med placebo.

京都大学 / 新制・課程博士 / 博士(社会健康医学) / 甲第24807号 / 社医博第131号 / 新制||社医||12(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 中山 健夫, 教授 山本 洋介, 教授 髙橋 良輔 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM