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  1. 1. SCREENING METHODS FOR ALZHEIMER DISEASE Presented By: Patel Dhwani J M Pharm , Pharmacology 1st year
  2. 2. INTRODUCTION • Alzheimer’s disease is an irreversible, progressive and neurodegenerative disease that slowly destroys memory and thinking skills, and ability to carry out simplest task. • It most often occurs in people over the age of 60-65. • Dementia is responsible for 60%. • Dementia is the loss of intellectual abilities, such as thinking, remembering and reasoning that is severe enough to interface with daily functioning. • In 1906 a German physician, Dr. Alois Alzheimer specifically identified a collection of brain cell abnormalities as a disease.
  3. 3. EPIDEMIOLOGY OF ALZHEIMER’S DISEASE • According to the Dementia in India 2020 report an estimated 5.3 million Indians aged >60 years had dementia in 2020, and this number is projected to exceed 14 million by 2050. • The number of Americans living with Alzheimer's is growing — and growing fast. • More than 6 million Americans of all ages have Alzheimer's. • An estimated 6.5 million Americans age 65 and older are living with Alzheimer's in 2022. Seventy- three percent are age 75 or older. • About 1 in 9 age 65 and older (10.7%) has Alzheimer's. • Almost two-thirds of Americans with Alzheimer's are women. • Worldwide, around 55 million people have dementia, with over 60% living in low- and middle- income countries. 75-84 years 85+ years 27% 37% 36% 65-74 years 2022 Alzheimer’s disease facts and figures-2022- Alzheimer’s & Dementia - Wiley Online Library
  4. 4. Symptoms
  5. 5. PATHOPHYSIOLOGY • Alzheimer’s disease is associated with brain shrinkage and localized loss of neurons, mainly in the hippocampus and basal forebrain. • The loss of cholinergic neuron in the hippocampus and frontal cortex is a feature of the disease and is thought to underline the cognitive deficit and loss of short term that occurs in AD. • Two microscopic features are characteristics of the disease, namely extracellular Amyloid plaques, consisting of extracellular deposits of β amyloid protein and intra neuronal neurofibrillary tangles, comprising filaments of a phosphorylated form of a microtubule associated Tau protein. • Hyperphosphorylated Tau and Aβ act synergistically to cause neurodegeneration.
  6. 6. • The cause of AD is considered to be the senile plaque (SP) formed by amyloid beta (Aβ) and neurofibrillary tangles (NFTs) composed of phosphorylated tau protein, in the hippocampus. • This leads to progressive cortical cell loss and cortical atrophy. HISTOPATHOLOGY
  7. 7. Drugs used in Alzheimer’s Disease
  8. 8. In- Vitro Screening Models 1. Inhibition of Acetylcholinesterase Activity in Rat striatum. 2. Inhibition of Butyrylcholinesterase Activity in Human serum. 3. Molecular forms of Acetylcholinesterase from Rat Frontal cortex and striatum. 4. Release of 3[H] Ach and other transmitters from Rat Brain Slices. 5. Ex-Vivo Cholinesterase Inhibition. 6. Stimulation of Phosphatidylinositol Turnover in Rat Brain Slices. 7. 3[H] Oxotremorine-M Binding to Muscarinic Cholinergic Receptors in Rat forebrain. 8. Uncompetitive NMDA Receptor Antagonism. In- Vivo Screening Models 1. Step Through 2. Up hill Avoidance 3. Elevated Plus Maze (EPM) 4. Step Down Method 5. Morris Water Maze Test 6. Visual Discrimination 7. Spatial Habituation Learning 8. Long-term potentiation in Hippocampal Slices. 9. Scopolamine induced amnesia in Mice. Screening Methods
  9. 9. 1. Inhibition of Acetylcholine-Esterase Activity in Rat Striatum. Purpose: • The purpose of this assay is to screen the drugs foe the inhibition of acetylcholine-esterase activity. • Inhibitors of this enzyme can be useful for the treatment of Alzheimer’s disease. • It is generally accepted that the physiological role of AchE is the rapid hydrolysis and inactivation of Acetylcholine. Procedure 1. 0.05M Phosphate buffer, pH 7.2 2. Substrate in buffer [ 198mg Acetylcholine chloride (10mM)] q.s to 100mL with 0.05M NaH2PO4 3. DTNB in buffer (19.8mg 5,5-dithiobisnitrobenzoic acid) q.s to 100mL with 0.05M NaH2PO4 4. A 2mM stock solution of the test drug is made up in a suitable solvent and q.s. to volume with 0.5mM DTNB. 5. Drugs are serially diluted (1:10) such that the final concentration is 10-4M and screened for activity.
  10. 10. Tissue Preparation 1. Male Wistar rats are decipitated 2. Brains are rapidly removed, corpora striata dissected free. 3. Weighed and homogenized in 19mL volume of 0.05M NaH2PO4 using Potter- Elvejhem homoginizer 4. A 25µL aliquot of this suspension is added to 1mL of the vehicle or various concentrations of the test drug. 5. Re-incubated for 10min at 37ºC.
  11. 11. Assay • Enzyme activity is measured with the Beckman DU-50 spectrophotometer. • Reagents are added to the blank and sample cuvettes as follows: • Blank: 0.8mL PO4 buffer/DTNB 0.8mL PO4 buffer/Substrate • Control: 0.8mL PO4 buffer/DTNB/ Enzyme 0.8mL PO4 buffer/Substrate • Drug: 0.8mL PO4 buffer/DTNB/Drug/Enzyme 0.8mL PO4 buffer/Substrate
  12. 12. Evaluation • For IC50 determination: • Substrate concentration is 10mM diluted 1:2 in an assay yielding a final concentration of 5mM. • DTNB concentration is 0.5mM yielding 0.25mM final concentration. • % Inhibition = Slope Control – Slope Drug 100 Slope Control
  13. 13. 2. Elevated Plus Maze Method  Purpose • Spatial discrimination task for rodents that has been extensively used in learning and memory studies, and that has been served as most important theories on the role of the hippocampus. • The maze allows the study of spatial reference and working memory process in the rats.
  14. 14. Procedure • The rats weighing around 200-250gm are housed in pairs for 10 days prior to testing. • Test drug administered 30minutes prior to experiment by i.p route. • The rat is then placed at the center of the maze facing one of the enclosed arm. • Animal is allowed to explore the maze for 5 minutes. • Observation is done from the adjacent room via remote TV camera.
  15. 15. Parameters Measured during 5 minutes of Observation: • Time spent in the open arm • Enteries into the open arm • Time spent in the closed arm • Enteries into the closed arm • Total arm enteries.
  16. 16. Case Study • A case of 58 year old white woman with the 2 year history of repetitiveness, memory loss, and executive function loss. • Neurocognitive assessment at the first clinic visit revealed a Mini Mental State Examination (MMSE) score of 14/30, poor verbal fluency patient was able to produce only 5 animal as well as poor visuospatial and executive skills. • After treatment with a cholinesterase inhibitor, her MMSE improved to 18/30, tested 15 months later with stability in function. Verbal fluency improved marginally with 7 animals. After an additional 18 months, function and cognition declined (MMSE=13/30) so memantine was added. • She died at age 63 of pneumonia. An autopsy was performed confirming the cause of death and her diagnosis of AD, showing numerous plaques and tangles with congophilic amyloid angiopathy. In addition, there was prominent Lewy Body pathology noted in the amygdala. • Various genetic mutations of the presenilin 1 (PSEN1) and presenilin 2 (PSEN2) as well as the amyloid precursor protein (APP) gene have been implicated. Mutations of PSEN1 and PSEN2 alter γ-secretase enzyme that cleaves APP resulting in increase in the relative amount of the more amyloidogenic Aβ42 that is produced.
  17. 17. Thank You

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