Screening models of alzheimer’s disease

1. Dr. APJ ABDUL KALAM TECHNICAL UNIVERSITY
(Formerly Uttar Pradesh Technical University) LUCKNOW
Noida Institute of Engineering & Technology
(Pharmacy Institute)
Project Title : Screening Models Of Alzheimer’s Disease
Submitted To:
Dr. Saumya Das
Associate Professor
Noida Institute of Engineering &Technology
(Pharmacy Institute)
Greater Noida
Submitted By:
Shilajit Das
M. Pharm(Pharmacology)
Noida Institute of Engineering &Technology
(Pharmacy Institute) Greater Noida

2. Alzheimer’s Disease - Introduction
▪ Alzheimer's disease (AD) is the most common neurodegenerative disease which has
features of progressive impairments in memory, behavior and cognition and can lead
to death.
▪ In addition to the financial burden of AD on health care system, the disease has
powerful emotional impact on caregivers and families of those affected.
Symptoms of AD is:
▪ Progressive dementia
▪ Loss of memory
▪ Cognitive decline
▪ Impairment of judgment
▪ Changes in personality

3. Drugs Used In Alzheimer’s Disease
Cholinesterase inhibitors
▪ Donepezil : Dose :- 5mg qd – 10mg qd
▪ Galantamine : Dose :- 8mg qd – 24mg qd
▪ Rivastigmine : Dose :- 4.6mg qd – 9.5mg qd
NMDA Receptor Antagonist
▪ Memantine : Dose :- 5mg qd – 10mg qd
NMDA= N-methyl-D-aspartate
Source: National Institute on Aging: Alzheimer's disease medications. November 2008. NIH Publication No
08-3431 Avalable at http://www.nia.nih.gov Alzheimers/Publications/medicationsts.htm. Accessed July 242009

4. Screening Method - In Vitro
1. Inhibition of acetylcholine-esterase activity in rat striatum.
Purpose and Rationale:
• To screen drugs for inhibition of acetylcholine-esterase activity..
• As it is generally accepted that the physiological role of ACHE is rapid hydrolysis and
inactivation of Ach. .
• Thus, inhibitor of AChE show marked cholinomimetic effects in cholinergically-innervated
effectors organs.
• Recent studies have suggested that AChE inhibitors may be beneficial for the treatment of
AD.

5. Screening Method- In Vitro
Procedure:
1. Reagents:
• 0.05 M Phosphate buffer, ph 7.2
• Substrate in buffer(198 mg acetylthiocholinechloride)
• DTNB in buffer (19.8 mg 5,5-dithiobisnitrobenzoicacid )(0.5mm)
• A 2mM stock sol. of test drug is made up in a suitable solvent and q.s to volume with 0.5mM
DTNB. Drugs are serially diluted(1:10) such that the final conc. in cuvette is 10-4 M and screened
for the activity.

6. Tissue prepration:-
It is reincubated for 10 min at 37°c.
25μl Aliquot of this suspension is added to 1ml of the vehicle and various concentration of test drug are prepared.
Then they are weighed and homogenized in 19 volumes (approx. 7 mg protein/ml) of 0.05 m nah2 po4, ph 7.2.
The brains are rapidly removed, the corpora striata is dissected freely.
They are decapitated
Rats weighing 150-200g are taken

7. Evaluation
• For IC 50 determinations Substrate conc. is 10 mM.diluted 1:2 in an assay yielding a
final conc. of 5 mm.
• DTNB conc. is 0.5 mM yielding 0.25 mM final conc.
% Inhibition = slope control - slope test × 100
slope control
• IC 50 values are calculated from log-probit analysis.

8. Inhibition of butyrylcholine esterase activity in
human serum
• Purpose and Rationale:
• This method is used in conjuction with the acetylcholine-esterase assay to determine
the enzyme selectivity of various cholinesterase inhibitors. •
• Butyrylcholine-esterase preferentially hydrolyses butyrylcholine..
• This enzyme is found in highest amount in serum, but its physiological role is not
known..
• Ethopropazine and Tetra-Isopropyl Pyrophosphoramide (ISO-OMPA) are Selective
inhibitors of butyrylcholinesterase.

9. Procedure
Reagents:
• 0.05 M Phosphate buffer, ph 7.2
• Substrate in buffer(225.8 mg s- butyrylthiocholinechloride)
• DTNB in buffer (19.8 mg 5,5-dithiobisnitrobenzoic acid) (0.5mm)
• A 2mM stock sol. of test drug is made up in a suitable solvent and q.s to volume
with 0.5mM DTNB. Drugs are serially diluted (1:10) such that determined from the
inhibitory activity of subsequent conc.

10. Enzyme preparation:
Pre-incubate for 10min at 37°c.
Added to 1ml of the vehicle & various conc. Of test drug are prepared
25 ml aliquot of this suspension
A vial of lyophilized human serum is reconstituted in 3ml of distilled water

11. Evaluation
• For IC 50 determinations Substrate conc. is 10 mM diluted 1:2 in an assay yielding a
final conc. of 5 mm.
• DTNB conc. is 0.5 mM yielding 0.25 mM final conc.
% Inhibition = slope control - slope test × 100
slope control
• IC 50 values are calculated from log-probit analysis.

12. Screening Method - In Vivo
Some of the in-vivo methods are:
Inhibitory(passive) avoidance:
• Step-down
• Step-through
• Two-compartment test
• Up-hill avoidance
• Trail-to-criteria inhibitory avoidance
• Scopolamine-induced amnesia in mice
• Memory impairment by basal forebrain lesions in rat•
• Ischemia-induced amnesia in gerbis
• Cognitive deficit on chronic low dose MPTP-treated monkeys
Active avoidance:
• Runway avoidance

13. 1. Step Down
Purpose and Rationale:
• An animal(mouse or rat) in an open spends most of the time close to the walls and in
corners..
• When placed on an elevated platform in the centre of a rectangular compartment, it steps
down almost immediately to the floor to explore the encloser and to approach the wall..
• This technique is employed in different modifications.

14. Procedure
Requirements:
• Mice or rats of either sex are used.
• A rectangular box (50x50) with electrifiable grid floor 35 fits over the block.
• Grid floor is connected to shock device.
A typical paradigm consists of :
• Familiarization
• Learning
• Retention test
Evaluation:
• The time of descent during the learning phase and the time during the retention test is measured.
• A prolongation of the step-down latency is defined as learning.

15. Procedure
Familiarization:
• Animal is placed on the
platform
• Released after raising the
cylinder
• Latency to descend is
measured
• After 10 seconds of
expolaration, it is returned to
the home cage
Learning
• Immediately the animal has
descended from the platform
• An avoidable shock is applied
(foot shock: 50Hz: 1.5 mA; 1
sec)
• Animal is returned to the
home cage
Retension test:
• 24 hrs after the learning trail
• The animal is again placed on
the platform
• step-down latency is measured
• The test is finished when the
animal steps down or remain
on the platform(cut-off time:
60 sec)

16. 2. Step through method
PURPOSE AND RATIONALE
This test uses normal behavior of mice and rats.
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.
The standard technique was developed for mice by Jarvik and Kopp
(1967) and modified for rats by King and Glasser (1970)

17. 2. 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 anacquisition trial followed by a retention trial
24 h later. In the acquisition trial the animal is placed in the illuminated
compartment at a maximal distance from the guillotine door, and the
latency to enter the dark compartment is measured
• Animals that do not step through the door within a cut-off time: 90 s
(mice) or 180 s (rats) are not used.

18. 2. Procedure
• Immediately after the animal enters the dark compartment, the door is
shut automatically and an unavoidable footshock (Footshock: 1 mA; 1 s-
mice; 1.5 mA; 2 s-rat) is delivered.
• The animal is then quickly removed (within 10 s) from the apparatus and
put back into its home cage.

19. 2. 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 stepthrough latencies is specific to
theexperimental situation.
• An increase of the step-through latency is defined as learning.

20. 3. 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.

21. 2. Procedure
• Hele of Dorn sex mice 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 tailelectrode 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

22. 2. Procedure
• During baseline-trials the animal's latency to make a 180° turn and
initiate the first climbing response is measured. Thereafter the animal is
returned to its home cage.
• During the experimental trials the latencies are measured and
additionally a tail-shock (1.5 or 2 mA) was administered contingent on
the first climbing response after the 180° turn.
• Immediately after the shock the animal is placed in its home cage. Retest
is performed 24 h later.

23. 2. Evaluation
• The latencies are measured.
Critical assessment of themethod:-
• The up-hill avoidance technique promises to provide a useful addition to
the existing arsenal of inhibitory (passive) avoidance methods.
• Its most obvious advantage is that it can be administered to animals
debilitated in sensory motor coordination by pharmacological or surgical
treatments.

24. 4. Scopolamine-induced amnesia in mice
Purpose and Rationale:
• The administration of anti- muscuranic agent scopolamine to young human
volunteers produces transient memory deficits..
• Similarly, scopolamine impairs memory retention when given to mice.
• The ability of different cholinergic drug agonist to reverse the amnesic affects of
scopolamine is now well documented in animal and human volunteers.

25. Procedure:
• The scopolamine test is performed in groups of 10 male NMRI mice weighing 26-32
g in one trail.
• Five min after i.p administration of 3mg/kg ofscopolamine hydrobromide.
• Each mouse is placed invidually in bright part of two chambered apparatus.
• After brief orientation period, mouse enters the secondor darker chamber.
• Once inside second chamber ,the doors are closed to avoid escape.
• A 1 MA, 1-sec foot shock is applied through grid floor. The mouse then return to the
home cage.

26. Procedure:
• 24 hrs later, testing is performed by placing the animal again in the bright chamber.
• The latency in entering the dark chamber within 5 min test session is measured
electronically.
• Whereas, untreated control animals enter the darker chamber in second trail with the
latency of about 250sec.
• Treatment with scopolamine reduces the latency to 50 sec.
• Test compounds are administered 90 min before training.
• The prolonged latency indicates that animal remembers that it has been punished and,
therefore, avoids darker chamber.

27. Evaluation
• After treatment with various doses of test drug latencies obtained were expressed as
% latencies.
• In some cases, straight dose-response curve isobtained whereas with other drugs
inverse U-shapeddose responses are observed.

28. References
▪ http://www.medicalnewstoday.com/articles/159442.ph
▪ Drug discovery and evaluation : pharmacological assays / H. Gerhard Vogel (ed.).-- 2nd edition
Pg No.599-601,619,623. Spain:Elsevier
▪ Alzheimer's Association www.alz.org
▪ Alzheimer's Disease Education and Referral(ADEAR) Center www.alzheimers.org
▪ Module 4481: Supporting the person with Dementia. TAFE NSW.
▪ National Institute on Ageing : http://www.nia.nih.gov
▪ http://en.wikipedia.org/wiki/Mementine http://en.wikipedia.org/wiki/Mementine
▪ Hospital and Clinical Pharmacy. Randhir Singha Dahiya and Dinesh Luther, Nirmala Prakashan
▪ Animal Models of Alzheimer Disease Frank M. LaFerla and Kim N. Green Institute for
Memory Impairments and Neurological Disorders, Department of Neurobiology and Behavior,
University of California, Irvine, Irvine, California 92697-4545.
▪ Drug Discovery and Evaluation Pharmacological Assays Co-Editors:Wolfgang H.Vogel
Bernward A. Schölkens Jürgen Sandow GünterMüller Wolfgang F. Vogel Second Edition.