The document discusses preclinical studies on anti-parkinsonian drugs, covering the etiology, pathophysiology, and classification of Parkinson's disease (PD), as well as various evaluation methods such as in vivo and in vitro models. It details different testing procedures using animal models to assess the efficacy of anti-parkinsonian treatments, including the MPTP model and other behavioral tests. The document emphasizes the neuroprotective effects of certain test drugs against dopaminergic cell degeneration caused by neurotoxins.

1. 1
PRECLINICAL STUDY OF
ANTI -PARKINSONIAN DRUGS
Presented by:
SANDIP CHAUDHARI
M. PHARM (1St
SEM)
Department of Pharmacology

2. 2
Contents
 Introduction
 Etiology
 Pathophysiology
 Classification
 Mechanism of action
 In vivo preclinical evaluation
 In vitro preclinical evaluation

3. 3
Introduction (Rang & Dale,2002, Richard ,A.H. 2000)
Parkinson's disease (PD) is a neurodegenerative movement
disorder characterised by progressive loss of dopaminergic neuron
in substantia nigra and depletion of the neurotransmitter
Dopamine in the striatum.
image
 Features
 Hypokinetic movement
 Akinetic- Difficulty in initiating movements & decreased
spontaneous movements.
 Bradykinesia – Slowness of movement.
 Decreased associated movements- Expressionless face or mask
face.

4. 4
 Hyperkinetic movements
 Rigidity – The limbs offer resistance to passive bending
throughout the movement.
 Tremor – Observed only at rest.
 Festinant gait- Trying to catch the center of gravity.

5. 5
Famous Faces of Parkinson
Michael J. Fox Muhammad Ali
Katharine Hepburn
Pope John Paul II Johnny Cash
Mao Tse Tung

6. 6
 Etiology
(Tripathi,2004)
 Idiopathic
 Drugs
 MPTP
 Genetic

7. 7
Pathophysiology (Goodmann & Gilmann,1996)

8. DA
ACH
PD due to DA
PD due to ACH
LevodopaInhibitory
Excitatory
Central anti
cholinergics drugs
8
(Frongois, W. et.al. 2003)

9. 9

10. 10
 Classification: (TripathiK.D,2004)
 Dopamine analogues: Levodopa
 Peripheral deoxy decarboxylase inhibitor: Carbidopa, benserazide
 COMT- inhibitors: Tolcapone, Entacapone
 MAO-B inhibitors: Selegiline, Clorgyline
 Dopamine agonists: Rosipirole , Pergolide
 Central anti- cholinergics: Benzotropine ,biperiden

11. 11

12. IN VIVO MODEL
 MPTP model in animals
A. MPTP model in mice
B. MPTP model in monkey
 Circling behavior test
 Elevation body swing test
 Reserpine antagonism
 Tremorine & Oxatremorine antagonism
 Specific test for evaluating locomotor activity
A. Skilled paw reaching test
B. Stepping test C. Gait Analysis 12

13.  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 -1.
 Most popular MPTP model :
1. MPTP model in mice.
2.MPTP model in monkey.
13
MPTP Model in animal (Von Bohlen,2006)

14. MPTP model in monkey s(Vogel, H .G. et al 2002)
 Principle:
N-MPTP
14
Partial damage to
basal ganglia
PD like symptom
DA precursor
L-dopa
Reversed
leads to
i.v.

15.  Requirements :
 Animal - Rhesus monkey (5-8 kg).
 Drug - N-MPTP up to 10-18 mg/kg i.v. for 5-8 days.
Test drug
15

16.  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
16

17.  Evaluation :
 The severity is rated by using scale of 0 (normal ) to 17 (max)
Observation Scoring
(1) Movement
Normal
Reduced
Sleepy
0
1
2
(2) Checking movement
Present
Reduced
Absent
0
1
2
17

18. Observation Scoring
(3) Attention& blinking
Normal
Abnormal
0
1
(4) Balance & co-ordination
Normal
Impaired
Unstable
Falls
0
1
2
3
(5) Vocalization
Normal
Reduced
Absent
0
1
2
18

19. MPTP model in mice (Wei, Zhang, et.al,2004)
 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.
19

20.  Requirements:
 Animal - mice-wild strain (C57BL/6J).
mice-null strain.
 Drug - MPTP (15mg free base/kg) s.c.
& test drug
20

21.  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
21

22. Striatal tissue are rapidly dissected
22
Striatal cell viability
estimation
NO release estimate by
assays
ROS estimate by
Fluorescence assays

23.  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.
23

24. Circling Behavior in rats (Bracha, H.S., et.al, 1986)
 Principle :
 An imbalance of dopaminergic activity within the basal
ganglia is associated with markedly asymmetric
circling behavior (Rotation turning) which measured by
Rota meter.
 This model is used in drug induced rotating behavior
and understanding of extra pyramidal disorder & of
their treatment by dopaminergic agents.
24

25.  Requirements:
 Animal - Sprague-Dawely rats(150-175gm)
 Dose - 6-OHDA(8µg in 4µl of 0.2mg/ml ascorbic acid in saline )
& test drug
 Apparatus – Rotameter
(www. harvardapparatus.com)
25

26.  Procedure:
Female Sprague Drawley rats are taken
Rats are anesthetized with Pentobarbital
(60mg/kg)
Head is placed in stereotaxic device (www.supertech.com)
Stereotaxically lesioned in left substantia nigra
(www.jove.com)
6-OHDA are injected into left SNr of rats
26

27.  L-desipramine.HCl (25mg/kg) are injected 30 min.
prior to 6-OHDA injected.
 Rats are divided into groups:
 Control groups for base value of ipsilateral rotation
-2.5mg/kg of d-amphetamine injected i.p. to rats.
 Control groups for base value of contra lateral
rotation- 1mg/kg of Apomorphine injected i.p. to
rats.
27

28.  Further studied test group as compared to control group.
 No. of full turns( either ipsilateral Or contra lateral turning to
lesion) are recorded an automatic print out counter every 15
min. for one or two hr. session.
28

29. Observation :-
For ipsi-lateral turning :
- administer 2.5 mg/kg Amphetamine &
placed in circling chamber for 2 hour
For contra-lateral turning :
- administer 1mg/kg & placed in
circling chamber for 1hour
Test compound are given i.p. or s.c. & record reading with 15
min. interval.
Evaluation :-
% change of drug turns from control turns is recorded.
29

30. Elevation body swing test (Cesanov, B;1995)
 Principle:
 EBST measures asymmetrical motor behavior of
hemiparkinsonian animals in a drug free state & drug induced
state.
 Drug induced motor behavior widely used as-behavioral index of
hemi parkinsonian animals.
 High positive co-relations b/w swing & Apomorphine induced
rotational behavior.
30

31.  Requirements:
 Animal - Sprague Dawley rats
 Drug - 6-OHDA (8 mg in 4ml 0.9% saline containing
0.02% ascorbic acid).
Test drug
31

32.  Procedure:
40 rats are taken as test group
Anesthetised with sod. Pentobarbital(60mg/kg i.p.)
mounted in stereotaxic device
stereotaxically lesioned in left substantia nigra
6-OHDA solution are injected over 4min. & needle left in
place for an additional 5 min. before retraction.
7 days after lesion, behavioral testing is performed .
Remaining 24 animal served as-control group.
The animals are placed into a plexiglass box (40x40x35.5 cm.)
32

33. Sr. no. 15 sec 30 sec 45 sec 60 sec
LS RS LS RS LS RS LS RS
33
 Observation :
 A swing is recorded whenever the animal moves its head out
of vertical axis.
swing are counted for 60 sec. with interval of 15sec.

34. Evaluation:-
Σno. of swing towards left side
% left swing =
Σ L+R
Σno. of swing towards right side
% right swing =
Σ L+R
34

35. Reserpine antagonism (Salvador, H.L., et al, 1996)
 Principle:
 The ability of anti-cholinergic agents microinjected in
subthalamic nucleus to reduce Reserpine induce
muscular rigidity are assesed in rats.
 Electro myographical activity of gastrocnemiussoleus
muscle are used as- a parameter of muscular rigidity.
 Reserpine increases high cholinergic tone in
subthalamic nucleus result in increased muscular
rigidity.
35

36.  Requirements:
 Animal : male rats (Wistar strain 280-300 gm)
 Drug – Chloral hydrate (350mg/kg i.p.)
Reserpine (5mg/kg i.p.)
 Apparatus- photocell
36

37.  Procedure:
male rats are taken
reserpine (5mg/kg i.p.) injected to rats
30min. Prior to observation test compound is injected
animals are placed singly on to floor of Perspex container
(30x26x20 cm.) which situated on panlab proximally
sensor unit.
Horizontal movement are recorded for 10min.
rearing & grooming episodes are registered
37

38. Evaluation :-
Locomotors activity & grooming scores of test group is
compared with control group.

39. Tremorine & Oxotremorine Antagonism (Parmar N.S,2000)
 Principle:
Tremorine and Oxotremorine is a muscarinic receptor
agonist, producing imbalance between the level of Ach & DA,
so inducing a parkinsonism. Produces a sign like tremors, ataxia,
salivation, lacrimation & hypothermia.
The signs are antagonized by test drug.
39

40. Requirements
Animal- NMRI mice ( male) (18-22gm)
Group- 2, each contain 6-10 mice
Drug- Test/ std , & tremorine or
oxotremorine
Dose- 5 mg/kg of standard drug
0.5 mg/kg oxotremorine
Route- s.c. for oxotremorine
40

41.  Procedure-
6-10mice are taken
0.5mg/kg Oxotremorine s.c administered
After 1hr test drug were given orally
Tremor is scored after Oxotremorine dosage in 10 sec. observation
periods in every 15 min.
Salivation & Lacrimation are scored 15 & 30 min.
after Oxotremorine injection.
41

42.  SCORING:-
Tremors/ lacrimation Score
absent 0
slight 1
medium 2
severe 3
video
42

43.  Evaluation :-
1) Hyperthermia :-
Average value of temp. in both group – compared
statistically.
2) Tremor :-
The score for all animals in each group. at the 3
observation periods are summarized .
The numbers in the treated groups. are expressed as
% of the no. of the controlled group.
3) Salivation & lacrimation :-
The course for both groups. are summarized at the 2
observation periods & expressed as % of the no. of
control group.
43

44. Skilled paw reaching test (Tracy, D.F., et.al., 2002)
 Principle:
 This method used to evaluate symptoms and treatment
in rat by skilled reaching with fore paw for food.
 Unilateral DA depletion reduces success by
abnormalities in movements including changing in
posture, shortened reaches and loss of pronation and
spuniation.
44

45. 45

46.  Requirements:-
 Animal - long Evans rats (250-310gm)
 Dose - Des methyl Imipramine (25 mg/kg i.p.)
6-OHDA (2μl of 4mg/ml in 0.95% saline with
0.02% ascorbic acid)
 Equipment- Single pellet boxes (25x35x30cm)
Food tray boxes(10x18x10cm)
46

47.  Procedure:-
20 rats are taken
30minute before surgery, desmethylimipramine administered
(25mg/kg i.p.)
Rats anesthetized with pentabarbital(60mg/kg i.p.)
12 rats received 6-OHDA lesions but 8 rats not received
47

48. Scoring reaching success:-
Reaching performance are scored by counting misses and
successful reaches for each limb.
1. Scored as “reach”.
2. Scored as “hit”.
Success% = no. of reaches /no. of hit x100
48

49.  Reaching posture
• Two point scale
• Scored as 0
• Scored as 1
49

50. Stepping Test in Rats (Olsson M. et. al,1995)
 Principle:-
This model is clinically relevant to unilateral model
for parkinsonism akinesia.
The 6-OHDA lesion induced marked and long-
lasting impairments in the initiation of stepping movements
with the contra lateral paw which can be ameliorated by
application of drug.
 Requirement:-
Animal: Sprague Drawley rats
Dose: 6-OHDA (3.6μg/μl in 0.2 μg/ml Ascorbate saline)
test drug
50

51.  Procedure:-
 Rats were placed in a steriotaxic device
 6-OHDA injected into the right ascending mesostriatal
 Experimental setup for stepping test.
 Evaluation parameter:-
Initiation time, Stepping Time, Step length.
51

52.  Step length = Length of ramp / no. of steps
 Sequence of testing in right paw & followed by left paw
testing, repeated twice.
52

53. Gait analysis (Nancy ,J.S. 2006)
(Jeffrey M. Hausdorff, et al. 1998)
 Gait analysis is useful in objective assessment of
walking ability and identify causes for walking
abnormality in parkinsonism disease.
 The result of gait analysis is useful in determining best
course of treatment.
 Catwalk method is mostly used to analyze gait in lab.
Animal.
53

54. 54

55.  Catwalk parameters-
 Regularity index
 Phase – lag
 Phase – lag variability
 Phase – lag mismatch %
 Symmetrical gaits are divided into two groups-
 A. Lateral sequence ( LS) gaits
 B. Diagonal sequence(DS)gaits
55

56. Rotenone induced parkinsonism (Todd, b., et.al.,2002)
 Principle:-
Chronic systemic complex 1st
inhibition caused
by Rotenone exposure induces of parkinsonism in rats
including selective nigrostriatal dopanimergic
degeneration & formation of ubiquitin & α-synuclein
inclusion .
56

57. 57

58.  Materials & Method:-
 Cell culture – SK-N-MC neuroblastoma cell
 Culture medium –minimum essential medium
 Reagents –rotenone(5nm)
prim. Abs & sec. Abs
 Visualizing reagent -3,3-diaminobenzidine
 Apparatus – bright field microscope
58

59.  Procedure :
SK –N- MC neuroblastoma cells cultured in MEC medium
containing rotenone
cells are incubated in prim. Abs for 24 hr.& followed by sec.
Abs for 1hr
Added visualizing agent
Observed under bright field microscope.
59

60.  Evaluation :
After 4 weeks, chronic rotenone exposure leads to –
 Synuclein & Ubiquitin level increased
 Reduced glutathione level.
 Caused oxidative protein damage.
 Caused oxidative DNA damage.
 Increased apoptotic death.
60

61. References
 Pal,G.K., 2007, Medical physiology, Ahuja publishing house, 1, 869-
870.
 Tripathi, K.D. (2004) “Antiparkinsonian Drugs” , Essential of
Medical Pharmacology Jaypee Brother’s Medical Publisher’s ,
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 Robert, P.S., et.al, 1997, Pharmacology of antiparkinsonism agents,
American Journal of Pharmaceutical Science 61, 179-180
 Goodman & Gilman’s, 1996, Treatment of central nervous system
degenerative disorders, Mcgrawl Hill, 9, 506-512.
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62.  Rang, P.H. et al (2003) “Nuerodegenerative Disease” Pharmacology,
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 Richard, A.H.(2000) “Treatment of Parkinsons Disease”,
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 Wei, Zhang, et.al. (2004) Neuroprotective effect of drugs in the
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Journal express article
 Von Bohlen and Halbach , (2006), Animal model of Parkinsons
disease ,Neurogenerative Disease 313 -320
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63.  Heikktla, R.E., et.al., 1981, L-dopa induced circling nigral lesions rats,
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 www. harvardapparatus.com
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 Salvador, H.L., et al, (1996) Muscarinic antagonist microinjected into
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64.  www.supertech picture mouse
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65.  Roger.W.et al, “Parkinson ‘s Disease” Clinical Pharmacy &
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66. THANK YOU
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