preScreening models for antiparkinsonism

1. PRECLINICAL
SCREENING OF DRUG
OF PARKINSONISM
B y - k a h k e s h a s a m s h a d
M . P h a r m ( p h a r m a c o l o g y )
1 6 0 0 1 0 0 5 5 8 B
I n t e g r a l u n i v e r s i t y, L u c k n o w.

2. INTRODUCTION.
• It is a progressive neurodegenerative disorder .
• Their is a marked deficiency in the dopaminergic innervation of the basal ganglia
owing to degeneration of neurones in the substantia nigra of brain.
• Enhancement of dopaminergic transmission restores at least partially motor
function.
• The decrease in dopaminergic activity in the basal ganglia results in a relative
excess of cholinergic influence.
• Therefore, dopaminergic agonists, such as levodopa, a precursor of dopamine,
and cholinergic (muscarinic) antagonists can be combined in the treatment of
Parkinson’s disease.
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3. IN-VIVO SCREENING MODELS.
• Tremorine and oxytremorine antagonism.
• MPTP model in monkeys.
• Reserpine antagonism
• Elevated body swing test.
• Skilled paw reaching test in rats.
• Stepping test in rats.
• Circling behavior in nigrostriatal lesioned rats.
• Reserpine antagonism.
• Rotenone induced parkinsonism
• Transgenic animal models of Parkinson’s disease.
• Cell transplantation into lesioned animals.
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4. TREMORINE AND OXYTREMORINE ANTAGONISM
• PURPOSE AND RATIONALE
• The muscarinic agonists tremorine and oxytremorine induce parkinsonism- like signs such
as tremor, ataxia, spasticity, salivation, lacrimation and hypothermia.
• These signs are antagonized by anticholinergic drugs.
PROCEDURE.
Groups of 6-10 male NMRI mice weighing 18–22 g are used.
They are dosed orally with the test compound or the standard (5 mg/kg benzatropine
mesilate) 1 h prior the administration of 0.5 mg/kg oxotremorine s.c
Rectal temperature is measured before administration of the compound (basal value) and 1,
and 3 h after oxytremorine injection.
Tremor is scored after oxytremorine dosage in 10 s observation periods every 15 min for 1
h.
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5. • TREMOR SCORE
– Absent 0
– Slight 1
– Medium 2
– Severe 3
– Salivation and lacrimation are scored 15 and 30min after oxotremorine
injection.
– Absent 0
– Slight 1
– Medium 2
– Severe 3
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6. • EVALUATION :
• Hypothermia
• The differences of body temperature after 1, 2 and 3 h versus basal values are
summarized for each animal in the control group and the test groups. The average
values are compared statistically.
• Tremor
• the scores for all animals in each group at the 3 observation periods are summarized.
The numbers in the treated groups are expressed as percentage of the number of the
control group.
• Salivation and lacrimation.
• The scores for both symptoms for all animals in each group are summarized at the 2
observation periods.
• The numbers in the treated groups are expressed as percentage of the number of the
control group
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7. MPTP MODEL IN ANIMALS
Principle:
• MPTP acts as- a neurotoxin which preferentially affected dopaminergic cells in
substantia nigra par compacta .
• MPTP shows toxicity due to conversion into MPP+ by MAO enzyme.
• This ion acts by inhibiting the ET system of mitochondrial complex-
• Most popular MPTP model:
1. MPTP model in mice.
2. MPTP model in monkey.
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8. 1. MPTP model in monkeys.
• PRINCIPLE: N-MPTP
(I.V)
Partial damage to basal ganglia leads to
PD like syndrome.
DA precursor L-dopa
Reversed.
REQUIREMENTS:
• Animal- rhesus monkey (5-8 kg)
• Drug- N-MPTP up to 10-18 mg/kg i.v for 5-8 days. Test drug.
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9. • PROCEDURE
Take 8 rhesus monkey (5-8 kg) of either sex.
Administered the N-MPTP i.v. with cumulative dose up to 10-18 mg/kg for 5-8 days.
Produces PD like symptoms
Administered test drug
Symptoms are Evaluated
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10. Evaluation :
The severity is rated by using scale of 0 (normal ) to 17 ((max)
Observation scoring
(1) Movement
Normal 0
Reduced 1
Sleepy 2
(2) Checking movement
Present 0
Reduced 1
Absent 2
(3) Attention& blinking
Normal 0
Abnormal 1
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11. (4) Balance & co-ordination
Normal 0
Impaired 1
Unstable 2
Falls 3
(5) Vocalization
Normal 0
Reduced 1
Absent 2
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12. • MPTP model in mice
• Principle:
• Neuro protective effect of test drug measured in MPTP model in mice.
• SN area is especially rich in microglia activation release a variety of neurotoxic factors
like superoxide, NO, cytokines & eicosanoids.
• Test drug reduces NADPH oxidase activity at extracellular & intracellular level.
Requirements:
Animal - mice-wild strain (C57BL/6J).mice-null strain.
Drug - MPTP (15mg free base/kg) s.c & test drug
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13. • Procedure:
Take NADPH - Oxidase null & wild type mice
MPTP (15mg/kg) injected s.c to mice daily for 6-consecutive days
Then test drug injected to mice twice daily for first 6-days& then inject once daily for
remainder study
After 6-days of last MPTP injection, mice are killed
Striatal tissue are rapidly dissected.
Striatal cell viability estimation.
• NO release estimate by assays
• ROS estimate by Fluorescence assays
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14. • Evaluation:
• Test drug evaluated for showing neuro protective action .
• Reducing NADPH Oxidase activity in PHOX+/+ (wild strain) is present but in PHOX-/-
NADPH Oxidase are absent .
• Reduces MPTP induced production of superoxide free radicals extracellular level &
intracellular ROS.
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15. IN- VITRO METHODS.
• Experiment using rat strial slices.
• Dopamine stimulated adenyl cyclase activity.
• Culture of substantia Nigra.
• Inhibition of apoptosis in neuroblastoma SH-SY5Y cells.
• Radioligand binding studies for D1 and D2 dopamine receptor.
• In-vitro neuroprotective efficacy.
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16. DOPOMINE STUMILATED ADENYLY CYCLASE ACTIVITY
• Male sprague- dawley(150-250g) are decapitated & right & left striata are removed.
• Striatal tissue is homogenized by teflon homogenizer in chilled buffer containing
10mM imidazole, 2mM EDTA & 10% sucrose ph 7.3.
• Homogenate is centrifuged at thousand g for10min & supernatant is recentifuged at
27000g for20min.
• The pellet obtained is washed twice & suspended in 10mM imidazole, ph 7.3.
• Membrane protein is determined by bradfords method using bovine serum.
• Adenylyl cyclase activity is measured by calculating the conversion rate of (32p) ATP
(32p) cAMP
.
• The assay is perform in 250μl solution containing imidazole, mgcl2 papaverine
dithiothreitol, ATP
, GTP
, phspocreatine, creatine phosphokinase.
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17. • The reaction mixture is preincubated at 30c for 5min, the reaction is initiated by adding
membrane proteins and incubated for 10min.
• The reaction is terminated by adding stopping solution(ATP, SDS, cAMP). Formed (32p)
cAMP is separated from (32p) ATP by chromatography.
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