a topic of biological evaluation

1. Screening of anticholinesterases
Presentedby:-
Kalyani thombre

2. Anti-cholinesterses (anti-Ches) are the agents which
 Inhibit ChE.
 Protect Ach from hydrolysis.
 Produce cholinergic effects in vivo.
 Potentiate Ach both in vivo and in vitro.
Introduction

5.  In vitro inhibition of acetylcholine-esterase activity in
rat striatum
 In vitro inhibition of butyrylcholine-esterase activity in
human serum
 Ex-vivo cholinesterase inhibition
 Molecular forms of acetyl-cholinesterase from Rat
Frontal Cortex and Striatum
 Release of (3H) Ach and other transmitters from Rat
Brain Slices
 (3H) Oxotremorine –M Binding in the presence and
absense of Gpp(NH)p
In vitro methods

6.  Stimulation of Phosphatidylinositol Turnover in Rat Brain
Slices
 (3H) N-Methylcarbamylcholine Binding to Nicotinic
Cholinergic Receptors in Rat Frontal Cortex
 Uncompetitive NMDA Receptor Antagonism
 Secreation of Nerve Growth Factor by Cultured Neurons
/Astrogial Cells
 Inhibition of RespirTORY Burst in Microglial Cells
/Macrophages

7.  Inhibitory Avoidance Methods
A. Step down test in mice and rats
B. Step through test in rodents
C. Uphill avoidance test in rats
D. Scopalamine-induced amnesia in mice
E. Ibotenic acid-induced impairment of memory
 Active Avoidance Tests
A. Runway avoidance in rats and mice
B. Shuttle box avoidance test(two way shuttle box)
C. Jumping avoidance in one-way shuttle box
In vivo methods

8.  Discrimination learning
A. Spatial habituation learning
B. Spatial discrimination test in rodents
C. Spatial learning test in the radial arm maze
D. Spatial learning test in the water maze
E. Olfactory learning in rats
 Conditioned response
A. Conditioned nictitating membrane response in rabbits

9. In vitro inhibition of acetylcholine-esterase activity on 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.
 PROCEDURE
 Reagents:-1) 0.05M,phosphate buffer , PH 7.2
 A) 6.85g NaH2PO4.H2O/100ml distilled H2O
In vitro methods

10. c) Add a) to b)until pH reaches 7.2
d) Dilute 1:10
2.Substrate in buffer
a) 198 mg acetylthiocholine chloride(10mM)
b) Q.s.explain to 100ml with 0.05M NaH2PO4,Ph 7.2
3.DTAB in buffer
a. 19.8mg 5,5-dithiobisnitrobenzoic acid (DTAB) (0.05mM)
b. Q.s. to 100ml with 0.05M NaH2PO4,Ph 7.2
4. A 2mM stock solution of the test drug is made up in a suitable
solvent and q.s. to volume with 0.05mM DTAB. Drugs are
serially diluted (1:10) such that the final concentration is 10-4M
and screened for the activity.

11.  Tissue preparation
 Male wistar rats are decapitated , brains rapidly
removed,corpora striata dissected free,weighed and
homogenized in 19 volumes of 0.05M NaH2PO4,PH 7.2
using a Potter-ELVEJHEM homogeizer.
 A 25ul of aliquot of this suspension is added to 1ml of the
vehicle or various concentrations of the test drug and
reincubated for 10min at 37degree celcius.
 Assay
 Enzyme activity is measured with the Beckman DU-50
spectrophotometer.
 this method can be used for IC50determinations and for
measuring kinetic constants.

12.  Reagents are added to the blank and sample cuvettes as
follows:-
 Blank: 0.8ml PO4 buffer/DTAB
 0.8ml buffer/substrate
 Control: 0.8ml PO4buffer/DTAB/Enzyme
 0.8ml PO4 buffer/substrate
 Drug: 0.8ml PO4 buffer/DTAB/Drug/Enzyme
 0.8ml PO4buffer/Substrate
EVALUATION
For IC50 determinations: substrate concentration is 10mM
diluted 1:2 in an assay yeilding a final concentrationof
5mM.DTAB concentration yeilding 0.25mM final
concentration.
%inhibition= slope control-slope drug *100
slope control

13. PURPOSE AND RATIONALE
This assay can be used in conjunction with the acetylcholine-
esterase assay to determine the enzyme selectivity of various
cholinesterase inhibitors.
PROCEDURE
Reagents
1. 0.05M phosphate buffer,PH 7.2
a) 6.85g NaHPO4.H2O/100ml distilled H2O
b) 13.40g NaHPO4.H2O/100ml distilled H2O
c) Add (a) to (b) until PH reaches 7.2
d) Dilute 1:10
In vitro inhibition of Butyrylcholine-
Esterase Activity in Human serum

14. 2.Substrate in buffer
a) 225.8mg s-butyrylthiocholine chloride
b) Q.s. to 100ml with 0.05M phosphate buffer,PH 7.2
3.DTAB in buffer
a) 19.8mg 5,5-dithiobisnitrobenzoic acid(0.5mM)
b) Q.s.to 100ml with 0.05M phosphate buffer,PH 7.2
4. A 2mM stock solution of the test drug is made up in a
suitable solvent and q.s. to volume with 0.5mM DTAB.
Drugs are serially diluted(1:10) such that determined from
the inhibitory activity of subsequent concentrations.
Enzyme preparation:
A vial of lyophilized human serum is reconstituted in 3ml of
distilled water.
A 25ml aliquot of this suspension is added to 1ml of the
vehicle or various concentrations of the test drug and
preincubated for 10min at 37degree cel.

15.  EVALUATION
 For IC50 determinations:
 Substrate concentration is 10mM diluted 1:2 in assay
yeilding final concentration of 5mM.DTAB
concentration is 0.5mM yeilding 0.25mM final
concentration.
 % inhibition = slope control-slope drug *100
slope control

16.  Inhibitory (passive) avoidance
• One of the most common animal tests in memory research is
the inhibition to initiate activities or learned habits.
• “Passive avoidance” is usually employed to describe
experiments in which the animal learns to avoid noxious event
by suppressing a particular behaviour.
 Step-down avoidance
Purpose and rational
When placed on an elevated platform in the centre of
rectangular compartment , it steps down almost immediately to
the floor to explore the enclosure and to approach the wall.
In vivo methods

17.  PROCEDURE
 it consists of three phases
 1.familirization
 2.learning
 3.retention test
 EVALUATION
 The time of descent
During the learning phase
And the time during retention test is measured.

18. Step-through method
PURPOSE AND RATIONALE
When placed into a brightly illuminated space connected to a dark enclosure,they
rapidly enter the dark compartment and remain there.
PROCEDURE
The test animals are placed in
The illuminated compartment
At a maximal distance from the
guillotine door,and the latency
to enter in dark comartment is
Measured.
EVALUATION
the time to step through during
the learning phase is measured
And the time during the retention
test is measured.

19.  Drug discovery and evaluation by wolfgang H. Vogel
2nd edition
 Screening methods in pharmacology by
N.S.Parmar,Shiv Prakash ,4th edition
REFERENCES