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Screening Methods for Neurodegenarative Diseases by - Vinayak patil
1. Screening Methods For
Neurodegenerative Disease’s
(Parkinson and Alhzeimer)
Name :- Patil Vinayak Bhanudas.
Department :- (M-Pharm) Pharmacology.
Roll No :- 12.
College Name :- Dr. Vithalrao Vikhe Patil Foundation’s College Of
Pharmacy
Vilad Ghat, Ahmednagar.
Prof. H. J. Pagar
3. Neurodegeneration :-
Progressive loss or death of neuronal cell’s is known as Neurodegeneration.
a)Protein misfolding and aggregation.
(Abnormal
Conformation)
(Soluble)
(Insoluble
Aggregates)
Deposition
Neurotoxicity
4. b) Excitotoxicity :-
Glutamates activate NMDA,AMPA and metabotropic receptor’s
Activation of AMPA causes the depolarization (i.e influx of Na+) and activation of NMDA leads to influx of Ca++
The balance between Na+/Ca++ is maintain by the efflux mechanism and it carry out by mitochondria and endoplasmic
reticulum
Due to hyperactivity of Na+ and Ca++ channels the conc. of Ca++ increased beyond the limit
It lead to damage the mitochondria and reduce the ATP synthesis
Formation of reactive oxygen species
Damages the neuronal cells
5. c) Oxidative Stress
Oxidation of Dopamine by MAO-B and Aldehyde dehydrogenase
Formation of reactive hydroxyl free radical (*OH) in presence of ferrous iron
This free radical are quenched by glutathione and other mechanism
This quenched free radicals damages the Enzymes, Membrane lipids and DNA molecules
They leads to the Neuronal death
7. Parkinsonism :-
• Extrapyramidal motor disorder in which the imbalance occurs between
Acetyl choline and Dopamine
• Here Neurodegeneration occurs at substantia nigra in mid brain
• Parkinsonism is first described by the scientist James Parkinson in 1817
Symptom’s :-
• Hypokinesia (Reduction in voluntary movement)
• Muscle Rigidity
• Tremor
9. Classification :-
Parkinson disease does not completely cure only symptomatic treatment are given to
the patients
Drug’s acting on brain Dopaminergic system
1) Dopamine Precursor :-
L-Dopa
2) Peripheral Dopa – Decarboxylase inhibitor :-
Carbidopa
Benserazide
3) Dopaminergic Agonist :-
Bromocriptine
Ropinirole
4) MAO-B inhibitor :-
Selegiline
5) COMT inhibitor :-
Entacapone
Tolcapone
6) Dopamine facilitator :-
Amantadine
Drug acting on brain Cholinergic system :-
1) Central Anti-Cholinergic agents :-
Benztropine
Biperiden
Procyclidine
2) Anti-Histaminic agents :-
Orphenadrine
Promethazine
10. Screening Models :-
In vivo Methods :-
• Tremorine and Oxotremorine Antagonism
• MPTT Model
• Elevated Body Swing Test
• Stepping Test in Rat’s
In vitro Methods :-
• Experiment using rat strial slices
11. 1) Tremorine and Oxotremorine Antagonism :- ( In vivo )
The muscarinic agonist tremorine and oxotremorine induce the Parkinsonism like signs such as tremor, muscle
rigidity, salivation, hypothermia, etc. This symptoms are Antagonized by Anticholinergic agents.
Groups of 6-10 male mice weight 18-22 gm are used
Orally administered the test compound or standard ( 5 mg/kg Benzatropine) 1 hr prior the administration of
oxotremorine s.c (0.5 mg/kg)
Rectal temperature is measured before administration of compound (Basal value) and 1, 2, and 3 hr after
oxotremorine injection
Tremor is measured after administration of oxotremorine (observation period was 15 min for 1 hr
Procedure :-
Evaluation :-
• Body Temperature
• Tremors
• Salivation
12. The MPTP act as a neurotoxin which specifically act on the Dopaminergic neurons in substantia nigra.
MPTP MPP+
The MPP+ is a reactive species which damages the neuronal cells
Procedure :-
Take 8 healthy Monkey (5-8 kg) of either six
Administered the N-MPTP (i.v) of dose 10-18 mg/kg
Due to MPTP the parkinsonism like symptoms are generated
Administered the test drug
Observed the reduction in symptoms
MAO-B
Evaluation :- Movement
Attention And Blinking
Balance and Co-Ordination
13. Elevation of Body swing test :-
Procedure :-
40 Rat’s are taken as test group
Anesthesia given to them (Sod. Phenobarbital 60mg/kg) i.p and placed into
stereotaxic frame.
The 6-OHDA solution is inject over 4 min. The needle is left in that place for another
administration after 5 min.
After 7 days the test is to be performed
Administered the test sample and place the animal in plexiglass box.
The animal is tie to the tail at 2.5 cm above the surface
Then check the swing attempt.
Evaluation :- • Left swing
• Right swing
In this method 6-OHDA (6-Hydroxy Dopamine) is used as a reagent.
This 6-OHDA induces the hyperseynsitivity at post synaptic dopaminergic receptor.
14. Expreiment using Rat staital slices :-
In vitro method
Procedure :-
Take male Rat’s (150-250 gm) They are Decapitated. Then the skull is opened.
Right and Left straitum is removed and placed into ice-cold kreb’s solution.
This straitum is cut into 0.4 mm thick slices by using tissue chopper.
The slice is keep floating for 30 min in kred solution.
This slice is Labelled by incubating for 30 min at 37 c temp with dopamine and choline in presence of
pargyline chloride and ascorbic acid.
This labelled slice is then transferred into superfusion chamber and perfused with krebs solution at 37 centrifuge
with 0.5ml/min flow rate.
After completion of this process we obtain the stabilized superfusate.
15. Then the drug is administrated into that superfusate fluid
By using scintillation counting measure the radio-activity
This superfusate is then placed into buffer solution. The buffer system will reduce the
dopamine and acetyl choline reuptake.
16. Alzheimer Disease :-
• It is a Neurodegenerative ,chronic ,irreversible disease which affects the neuronal cells in the brain and
causes impairment of memory.
• In this disorder the patient are in complete vegetative state and the ability of respond, imagining,
remember and learning will be gradually reduced
• The Alzheimer disease is first described by Dr. Alois Alzheimer in 1906
• The loss of cholinergic neurons at the hippocam
basal forebrain and frontal cortex.
• Neuronal death is caused due to two mechanism
Extracellular deposition.
Intracellular deposition.
17. Classification :-
A) Cholinesterase inhibitor :-
Donepezil
Rivastigmine
Galantamine
B) Cholinergic activator :-
Tacrine
C) NMDA (Glutamate) antagonist :-
Memantine
D) Anti-Oxidants :-
Ginkgo biloba
Vitamin-K
Selegeline (MAO- B inhibitor)
E) Statins :-
Simvastatin
Pravastatin
18. Screening Models :-
In Vitro Models :-
In vitro inhibition of Acetylcholinesterase activity in Rat striatum
In vitro inhibition of Butyrylcholinesterase activity in human serum
In vivo models :-
Step down
Step through
)
19. In vitro inhibition of Acetylcholinesterase activity in rat striatum :-
Procedure :-
Regants :–
1) 0.05 M Phosphate buffer ph 7.2
2) Substrate in buffer
(198 mg acetylcholine chloride)
3)DTNB in buffer
(19.8 mg 5,5-dithiobisnitrobenzoic acid)
4) Test solution
Tissue Preparation :-
Male rat’s are decapitated, Brain are rapidly
removed, Corpora striata dissected free, Weight and homogenized in 19
volume of 0.05 M Nah2Po4 by using homogenizer. The 25 ul of this
suspension is added in 1 ml vehicle or in various conc. of test drug and re-
incubated for 10 min at 37 C
Assay :-
Enzyme activity is measured with Beckman DU50
Spectrophotometer, The IC50 is measured.
20. In vitro inhibition of Butyrylcholinesterase activity in human
serum
This in vitro assay is used to determine the Enzyme selectivity of cholinesterase inhibitor
Procedure :-
Regants :–
1) 0.05 M Phosphate buffer ph 7.2
2) Substrate in buffer
(225.8 mg Butyrylthiocholine chloride)
3)DTNB in buffer
(19.8 mg 5,5-dithiobisnitrobenzoic acid)
4) Test solution
Enzyme preparation :-
Lyophilized human serum, It reconstituted in 3 ml distilled water,
A 25 ul of this suspension is added into vehicle or in various concentration of test solution,
pre-incubated for 10 min at 37 C
Assay :-
The enzyme activity is measured by using Beckman DU50
Spectrophotometer. This method is used for measuring IC50.
21. In vivo method :-
Step down test :-
Procedure :-
Mice or rat’s of either sex is used. A rectangular box (50-50) with electrifiable floor and 35
cm fit’s over the block. The grid floor is connected to shock device which gives the foot shock.
1) Familiarization :-
The animal is placed on the platform and released after 10 sec. They try to move
from block to grid floor
2) Learning :-
Immediately after animal is move form block to grid floor the unavoidable foot shock is
applied (50 Hz) and animal is return to home cage.
3) Retention Test :-
After 24 hr of learning test the animal is placed on block and measured the step-
down latency period. The test is finished when the animal is step-down or remain on block.
