Screening Models of Alzheimers disease.pptx

Alzheimers disease
symptoms
pathophysiology
screening models - in-vitro and in-vivo models

1. Screening Models of Alzheimers NOIDA INSTITUTE OF ENGINEERING AND TECHNOLOGY (PHARMACY INSTITUTE) GREATER NOIDA Presented By: Archna Singh M.Pharm (Pharmacology) PTSM-1 Under Guidance: Dr. Saumya Das Professor (H.O.D. Pharmacology) NIET (Pharmacy Institute) Greater Noida

2. ALZHEIMER’S DISEASE Neurodegenerative disease or Aging Disease. Progressive impairment of memory and cognitive function. Development of senile plalgues = Neuronal Destruction Etiology = Destruction of nerve= Cholinergic= Ach Treatment= Ach level by Acetylcholinestrase

3. SYMPTOMS Poor Judgement Depression, Anxiety, Insomnia Misplacing objects

4. Pathophysiology Cholinergic Hypothesis Amyloid Hypothesis Tau Hypothesis Acetylcholine Plaques Beta- Amyloid This Photo by Unknown Author is licensed under CC BY-SA-NC

5. Screening Methods

6. In-Vitro Models Inhibition of Acetylcholinestrase Activity in Rat Striatum. 2. Inhibition of Butylcholinestrase Activity in Human Serum. 3. Molecular forms of Acetylcholinestrase from rat frontal cortex and striatum. 4. Release of [H3] Ach and other transmitters from Rat Brain Slices. 5. Ex-Vivo Cholinestrase Inhibition. 6. Stimulation of phosphatidylinositol Turnover in Rat Brain Slices. 7. Uncompetitive NMDA Receptor Antagonism.

7. Inhibition of Acetylcholinestrase Activity in Rat Striatum PURPOSE AND RATIONALE The purpose of this assay is to screen drugs for inhibition of acetylcholine-esterase activity. Inhibitors of this enzyme may 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. AChE inhibitors may also be beneficial in the treatment of Alzheimer’s dementia.

8. PROCEDURE Reagents 1. 0.05 M Phosphate buffer, pH 7.2 - 6.85 g NaH2PO4 · H2O/100 ml distilled H2O 2. Substrate in buffer -198 mg acetylthiocholine chloride (10 mM) 3. DTNB in buffer - 19.8 mg 5,5-dithiobisnitrobenzoic acid (DTNB) (0.5 mM) 4. A 2 mM stock solution of the test drug is made up in a suitable solvent and q.s . to volume with 0.5 Mm DTNB (reagent 3). Drugs are serially diluted (1 : 10) such that the final concentration (in cuvette) is 10–4 M and screened for activity. If active, IC50 values are determined from the inhibitory activity of subsequent concentrations.

9. Tissue Preparation Male Wistar rats are decapitated, brains rapidly re moved, corpora striata dissected free. Weighed and homogenized in 19 volumes (approximately 7 mg protein/ml) of 0.05 M NaH2PO4, pH 7.2 using a Potter- Elvejhem homogenizer ( Kontes , Vineland, NJ). A 25 µl aliquot of this suspension is added to 1 ml of the vehicle or various concentrations of the test drug and reincubated for 10 min at 37 °C.

10. Assay Enzyme activity is measured with the Beckman DU-50 spectrophotometer. This method can be used for IC50 determinations and for measuring kinetic constants. Reagents are added to the blank and sample cuvettes as follows: Blank: 0.8 ml PO4 buffer/DTNB 0.8 ml buffer/Substrate Control: 0.8 ml PO4 buffer/DTNB/Enzyme 0.8 ml PO4 buffer/Substrate Drug: 0.8 ml PO4 buffer/DTNB/Drug/Enzyme 0.8 ml PO4 buffer/Substrate Blank values are determined for each run to control for non-enzymatic hydrolysis of substrate and these values are automatically subtracted by the kindata program available on kinetics soft- pac module.

11. EVALUATION For IC50 determinations: Substrate concentration is 10 mM diluted 1 : 2 in an assay yielding a final concentration of 5 mM. DTNB concentration is 0.5 mM yielding 0.25 mM final concentration %Inhibition = slope control – slope drug slope control IC50 values are calculated from log-probit analysis. X 100

12. In-Vivo Methods

13. Step-Down Method Purpose and Rationale An animal (mouse or rat) in an open field spends most of the time close to the walls and in the corners. When placed on an elevated platform in the center of a rectangular compartment, it steps down almost immediately to the floor to explore the enclosure and to approach the wall.

14. Procedure Mice or rats of either sex are used. Training is carried out in 2 sessions Animal is placed on the elevated platform Animal steps down on to the grid ( innate exploratory behavior) Electric shock (15sec) is given on the grids Step down latency (SDL) is noted

15. EVALUATION The time of descent during the training phase and the time during retention test is measured. Prolongation of the step-down latency is defined as learning.

16. MODIFICATION Induce amnesia to animals. Different methods to do so- Electroconvulsive shock Scopalamine alcohol

17. STEP-THROUGH METHOD Purpose and Rationale This test uses normal behavior of rats and mice. These animals avoid bright light and prefer dim illumination. When placed into a brightly illuminated space connected to a dark enclosure, they rapidly enter the dark compartment and remain there.

18. Procedure Mice and rats of either sex are used. The test apparatus consists of a small chamber connected to a larger dark chamber via a guillotine door. The test animals are given an acquisition trail followed by a retention trail 24hr later. In the acquisition trail the animal is placed in the illuminated compartment at the maximal distance from the door, and latency to enter the dark compartment is measured. Animals that do not step through door within a cut-off time: 90 sec(mice) or 180sec(rats) are not used.

19. . Immediately after the animal enters the dark compartment, the door is shut automatically and footshock is delivered. The animal is then quickely removed (within 10sec) from the apparatus and put back into its home cage.

20. EVALUATION The time to step-through during the learning phase is measured and the time during the retention test is measured. In this test a prolongation of the step- through latencies is specific to the experimental situation. An increase of the step-through latency is defined as learning.

21. UP-HILL AVOIDANCE METHOD PURPOSE AND RATIONALE Many animal species exhibit a negative geotaxis, i . e. the tendency to orient and move towards the top when placed on a slanted surface. When placed on a tilted platform with head facing down-hill, rats and mice invariably turn around and move rapidly up the incline.

22. PROCEDURE Rats of both sex were used and maintained under standard conditions. The experimental apparatus is a 50 x 50 cm box with 35 cm high opaque plastic walls The box can be inclined at different angles. The floor consists of 10 mm diameter stainless steel grid bars placed 13 mm apart To deliver the tail-shock-a- tail electrode is constructed, consisting of a wire clip connected to a constant current shock source. The animal is first fitted with the tail-electrode and then placed onto the grid with its nose facing down .

23. . During baseline-trails the animal’s latency to make a 180 degree turn and initiate the first climbing response is measured. Thereafter the animal is returned to its home cage. During the experimental trails the latencies are measured and additionally a tail-shock (1.5 or 2 mA) is administered contingent on the first climbing response after the 180 degree turn. Immediately after the shock the animal is placed in its home cage. Retest is performed 24hr later.

24. EVALUATION The latencies are measured. CRITICAL ASSESSMENT OF THE METHOD The up-hill avoidance technique promises to provide a useful addition to the existing resource of inhibitory (passive) avoidance methods. Its most obvious advantage is that it can be administered to animals weakened in sensory motor coordination by pharmacological or surgical treatments.

25. RUNWAY AVOIDANCE Purpose and rationale A straightforward avoidance situation features a fixed aversive gradient which can be transversed by the animal. The shoc can bee avoided when the safe area is reached within the time allocated.

26. PROCEDURE Mice or rats of either sex are used and handled for several days before the experiment. The same box as used in step-through model can be used in this experiment. The apparatus is uniformly illuminated by an overhead light source. A loudspeaker, mounted 50cm above the start-box, serves for presenting the acoustic conditioned stimulus

27. . . The footshock is employed Animal is allowed to explore whole apparatus for 5 min. The guillotine door is then closed and the animal is closed and animal is placed into the light starting area. After 10sec the acoustic CS is applied and the door is opened. Shock is turned on after 5 sec. The procedure starts again. The training is continuous until the animal attains the criterion of 9 avoidance in 10 trails. The time needed to reach the safe area is measured.

28. EVALUATION The time animal required to reach the safe area on both days is measured. In addition, the number of errors (not reaching the safe area) is recorded.

29. REFERENCE H. Gerhard Vogel (Ed.) (2002) “Drug Discovery and Evaluation-Pharmacological Assays”, 2 nd edition. Springer-Verlag Berlin Heidelberg Publishers, New York, pp 619-626 Gupta SK. (2016), Drug Screening Method, 3 rd edition, Jaypee Brothers Medical Publishers (P) Ltd, pp 514-532 https://www.slideshare.net/roohna/screening-of-anti-alzheimers https://www.slideshare.net/DrxBurade/preclinical-screening-for-alzheimers https://www.slideshare.net/mohammadmuztaba/alzheimer-models