This document provides an overview of preclinical studies used to screen anti-parkinsonian drugs. It describes several in vivo and in vitro methods used, including the MPTP monkey model, reserpine antagonism in mice, and circling behavior in nigrostriatal lesioned rats. The goal of these preclinical studies is to evaluate potential new drugs for their ability to reduce Parkinson's disease motor symptoms before clinical trials in humans.

1. PRECLINICAL STUDY OF
ANTI -PARKINSONIAN
DRUGS
Submitted by HINNA HAMID
M Pharm 1st year (PHARMACOLOGY )
Department of Pharmaceutical sciences ,
UNIVERSITY OF KASHMIR,SRINAGAR.

2. CONTENTS :
 Introduction
 Pathophysiology
 Classification
 Pre Clinical Screening of Anti Parkinsonian Drugs.
o In vivo methods
o In vitro methods
 Bibliography.

3. INTRODUCTION
 Parkinson’s disease (PD) is a neurodegenerative
progressive disorder, mostly affecting older people,
first described by James Parkinson in 1817.
 Features
 Hypokinetic movement
 Akinetic- Difficulty in initiating movements &
decreased
 spontaneous movements.
 Bradykinesia - Slowness of movement.
 Decreased associated movements- Expressionless
face or like mask face.

4. TO BE CONTINUED :
 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. PATHOPHYSIOLOGY
 The most consistent lesion in PD is degeneration of
neurones in the substantia nigra pars compacta
(SN-PC) and the nigrostriatal (dopaminergic) tract.
 This results in deficiency of dopamine (DA) in the
striatum which controls muscle tone and
coordinates movements.
 An imbalance between dopaminergic (inhibitory)
and cholinergic (excitatory) system in the striatum
occurs giving rise to the motor defect. Though the
cholinergic system is not primarily affected, its
suppression (by anticholinergics) tends to restore
balance.

6. TO BE CONTINUED :

7. TO BE CONTINUED :
Normal vs parkinsonism

8. CLASSIFICATION
I. Drugs affecting brain dopaminergic system :
(a) Dopamine precursor : Levodopa (l-dopa)
(b) Peripheral decarboxylase inhibitors : Carbidopa,
Benserazide.
(c) Dopaminergic agonists: Bromocriptine, Ropinirole,
Pramipexole
(d) MAO-B inhibitor: Selegiline
(e) COMT inhibitors: Entacapone, Tolcapone
(f) Dopamine facilitator: Amantadine.

9. TO BE CONTINUED :
II. Drugs affecting brain cholinergic system
(a) Central anticholinergics: Trihexyphenidyl
(Benzhexol), Procyclidine, Biperiden.
(b) Antihistaminics : Orphenadrine, Promethazine.

10. METABOLIC PATHWAYS OF LEVODOPA IN
THE PERIPHERY AND THE BRAIN.

11. PRE CLINICAL SCREENING OF ANTI
PARKINSONIAN DRUGS.
 In vivo methods :-
 Tremorine and oxotremorine antagonism
 MPTP model in monkeys
 Reserpine antagonism
 Circling behavior in nigrostriatal lesioned rats
 Elevated body swing test
 Skilled paw reaching in rats
 Stepping test in rats

12. TO BE CONTINUED :
 In vivo methods :-
 Experiments using rat striatal slices
 Dopamine stimulated Adenylyl Cyclase activity
 Radioligand binding studies for Di and D2
Dopamine receptors
 Dopamine release from synaptosomes

13. TREMORINE AND OXOTREMORINE
ANTAGONISM
 PURPOSE AND RATIONALE :-
 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.

14.  PROCEDURE
01
• Groups of 6–10 male NMRI mice weighing 18–22
are used.
02
• They are dosed orally with the test compound or the
standard (5 mg/kg benzatropine mesilate) 1 prior the
administration of 0.5 mg/kg oxotremorine s.c.
03
• Rectal temperature is measured before
administration of the compound (basal value) and 1,
2 and 3 h after oxotremorine injection.

15. TO BE CONTINUED :
04
• Tremor is scored after oxotremorine dosage in
10s observation periods every 15 min for 1 h.
05
• Salivation & Lacrimation are scored 15 & 30
min after Oxotremorine injection.

16. TO BE CONTINUED :
Tremor Score
absent 0
slight 1
medium 2
severe 3

17. TO BE CONTINUED :
Salivation and
lacrimation
Score
Absent 0
Slight 1
medium 2
Severe 3

18. TO BE CONTINUED :
 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.

19. TO BE CONTINUED :
 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.

20. MPTP MODEL IN MONKEYS
PURPOSE AND RATIONALE
 N-MPTP (N-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine) has been shown to cause
symptoms of Parkinson’s disease in exposed
individuals.
 When administered to primates this compound
causes a partial destruction of basal ganglia and a
syndrome that resembles Parkinson’s disease.

21. PRINCIPLE :-

22. PROCEDURE
01
• Take 8 rhesus monkey (5-8 kg) of either
sex
02
• Administered the N-MPTP i.v. with
cumulative dose upto 10-18 mg/kg for 5-8
days.
• Produces PD like symptoms
03
• Administered test drug
• Symptoms are Evaluated

23. EVALUATION
 The severity of parkinsonian symptoms is rated by
trained observers using a scale of 0 (normal) to 17
(maximum severity) that assesses
01 ) movement
(0: normal;1: reduced; 2: sleepy),
02) checking movements
(0: present; 1: reduced; 2: absent),
03) attention and blinking
(0: normal; 1: abnormal),
04) vocalizations
(0: normal; 1: reduced; 2: absent).

24. TO BE CONTINUED :
05 ) balance and coordination
(0: normal; 1: impaired;
2: unstable; 3: falls),
06) Reactions
(0: normal; 1: reduced; 2: slow; 3: absent)
and
07 ) Posture
(0: normal;
1: abnormal trunk;
2: abnormal trunk and tail;
3: abnormal trunk, tail, and limbs;
4: flexed posture),

25. RESERPINE ANTAGONISM
PURPOSE AND RATIONALE
 Reserpine induces depletion of central
catecholamine stores.
 The sedative effect can be observed in mice shortly
after injection, followed by signs of eyelid ptosis,
hypokinesia, rigidity, catatonia, and immobility.
 These phenomena can be antagonized by
dopamine agonists.

26. PROCEDURE
01
• Male NMRI mice of either sex weighing
20–25 g are used
02
• They are injected intraperitoneally with
5 mg/kg reserpine and tested 24 h later
03
• Thirty min prior to observation the test
compounds are injected.

27. TO BE CONTINUED :
04
• The animals are placed singly onto the floor of
a Perspex container (30 × 26 cm, 20 cm high),
situated on a Panlab proximit on a sensor unit.
05
• Horizontal movements are recorded for 10 min
06
• Moreover, rearings and grooming episodes
are registered by an experienced observer.

28. EVALUATION
Locomotor activity and grooming scores of
drug treated animals are compared with
controls treated with reserpine and vehicle
only by analysis of variance.

29. CIRCLING BEHAVIOR IN NIGROSTRIATAL
LESIONED RATS
PURPOSE AND RATIONALE
 Unilateral lesion of the dopaminergic nigrostriatal
pathway in the rat by the neurotoxin 6-hydroxydopamine
(6OHDA) induces hypersensitivity of the postsynaptic
dopaminergic receptors in the striatum of the lesioned
side.
 The rats rotate in a direction towards the lesioned side
(ipsilateral) when an indirect acting compound such as
amphetamine is administered but to the opposite
direction (contralateral) when a direct acting dopamine
agonist, e.g., apomorphine, or the dopamine precursor
L-dopa is given.
 Therefore, this test can be used for the study of central
dopamine function and the evaluation of dopamine
antagonists and agonists, particularly the activity of
novel antiparkinsonian drugs.

30. PROCEDURE
01
• Male Wistar rats weighing 200–250 g at the time of
surgery are used.
• The are housed individually in a controlled
environment with free access to food and water.
02
• The animals are anaesthetized with sodium
pentobarbital.
• The head is placed in a stereotaxic device (DKI
900) and positioned according to the atlas of König
and Klippel.
03
• After a sagittal cut is made in the skin of the skull, a
2 mm wide hole is drilled with an electrical trepan
drill.
• Care is taken not to lesion the meninges.

31. TO BE CONTINUED :
04
• A 30 gauge stainless-steel cannula connected to a
Hamilton syringe is aimed at the anterior zona
compacta of the substantia nigra (coordinates
anterior 4.1, lateral 1.0 and dorso-ventral –2.5 from
instrument zero).
05
• A total of 8 μg of 6-OHDA in 4 γ/L of saline is
injected at a rate of 1 γ/L/min. After the intracranial
injection the wound is closed.
06
• The animal is allowed several weeks for recovery
and for development of the lesion.

32. TO BE CONTINUED :
07
• Specially constructed opaque plastic spheres
attached to solid state programming equipment
serve as test chambers.
08
• The number of full turns, either ipsilateral or
contralateral to the lesion, are recorded on an
automatic printout counter every 15 min for one- or
two-hours test sessions.
09
• To determine the control values for ipsilateral
turning, each subject is administered 2.5 mg/kg of d-
amphetamine and immediately placed in the circling
chamber for 2 h.

33. TO BE CONTINUED :
10
• Control values for contralateral circling are
determined by injecting apomorphine at 1 mg/kg
s.c. and recording the rat’s circling for 1 h.
11
• Test compounds are given i.p or sc. and the
animals placed into the circling chambers.
12
• Circling is recorded over a 1 h period.

34. TO BE CONTINUED :
EVALUATION
 Percent change of
drug turns from
control turns is
recorded.
 Using various doses
ED50 values can be
calculated.

35. ELEVATED BODY SWING TEST
PURPOSE AND RATIONALE
 Borlongan and Sanberg (1995) proposed the
elevated body swing test as a measure of
asymmetrical motor behavior of hemiparkinsonian
animals in a drug-free state.
 Drug induced motor behavior widely used as-
behavioral index of hemi parkinsonian animals.

36. PROCEDURE
01
• Male, 8-week-old Sprague Dawley rats are
anesthetized with sodium pentobarbital (60
mg/kg i.p.) and mounted in a Kopf
stereotaxic frame.
02
• They are lesioned by injection of 8 μg 6-
hydroxydopamine in 4 μl saline containing
0.02% ascorbic acid in the left substantia
nigra (AP – 5.0, ML + 1.5, DV – 8.0)
03
• The solution is injected over a 4 min period
and the needle left in place for an additional
5 min before retraction.

37. TO BE CONTINUED :
04
• Seven days after the lesion, behavioral
testing is performed.
05
• The animal is allowed to habituate in a
Plexiglas box and attain a neutral position
having all 4 paws on ground
06
• The rat is held about 2.5 cm from the base
of its tail and elevated 2.5 cm above the
surface on which it has been resting.

38. TO BE CONTINUED :
07
• A swing is recorded whenever the animal
moves its head out of the vertical axis to
either side.
08
• Before attempting an other swing, the
animal must return to the vertical position
for the next swing to be counted.
09
• Swings are counted for 60 s over four
consecutive 15 s segments.

39. TO BE CONTINUED :
10
• The total number of swings made to each
side is divided by the overall total number
of swings made to both sides to get
percentages of left and right swings.
11
• The criterion of biased swing is set at 70%
or higher
12
• At 30 and 45 s, 6-OHDA-lesioned rats
exhibit right biased swings of 70% or
higher compared to normal rats.

40. EVALUATION
 A two-way ANOVA is used to analyze swing
behavior data across the 15 s segments.

41. EVALUATION

42. SKILLED PAW REACHING IN RATS
PURPOSE AND RATIONALE
o This method used to evaluate symptoms and
treatment in rat by skilled reaching with fore paw for
food.
 Skilled paw reaching test is used as a model of
Parkinson’s disease in the rat.
 Unilateral injection of 6-OHDA into the medial
forebrain bundle results in an impairment of paw
reaching on both sides which can be ameliorated
by drug treatment or transplantation of a nigral cell
suspension.

43. PROCEDURE
 Apparatus
 The apparatus consists of a clear Perspex chamber with
a hinged lid.
 A narrower compartment with a central platform running
along its length, creating a trough on either side, is
connected to the chamber.
 The narrowness of the side compartment prevents rats
from turning around, so they can use only their left paw
for reaching into the left trough and their right paw for
reaching into the right trough.
 A removable double staircase is inserted into the end of
the box, sliding into the troughs on either side of the
central platform.
 Each of the steps of the staircase contains a small well,
and two 45 mg saccharin-flavored pellets are placed in
each well.

44. APPARATUS :-

45. TO BE CONTINUED :
 Learning procedure
 The week before the start of the training period, the rats
are deprived of food and their body weight is stabilized
at 85% of the weight of non-deprived rats.
 At the same time, they are gently manipulated and
familiarized with the appetitive saccharin-flavored
pellets.
 The animals then begin to learn the paw reaching task.
 For four weeks they are placed in the test boxes once
per day for 10–15 min.
 The number of pellets eaten during the test period
indicates the rat’s success in grasping and retrieving the
pellets;

46. TO BE CONTINUED :
o the number of steps from which pellets have been
removed provides an index of the attempts to reach the
food and how far the rat can reach;
o the number of missed pellets remaining at the end of
the test on the floor of the side compartment indicates a
lack of sensorimotor coordination in grasping and
retrieving the pellets.
o In addition to these three parameters, it is noted which
forepaw the rat used for the first movement to reach the
pellet on each test day.
o A first choice score of +1 corresponds to the paw
contralateral to the lesion, a score of –1, to the paw
ipsilateral to the lesion.
o Because rodents exhibit a “pawedness”, it must be
noted whether there is a preference for one paw.

47. TO BE CONTINUED :
 Lesions
 The mesotelencephalic system is lesioned by a
stereotaxic unilateral injection of 6-OHDA into the medial
forebrain bundle under equithesin anesthesia.
 6-OHDA is injected in a volume of 1.5 μl and at a
concentration of 4 μg/μl 0.9% saline and 0.01% ascorbic
acid twice over 3 min via a 30-gauge stainless steel
cannula.
 Following each injection, the cannula is left in place for
an additional 4 min to allow the diffusion of the
neurotoxin away from the injection site.
 The shamoperated group receives sham lesions by
identical injection of ascorbate-saline solution alone.

48. TO BE CONTINUED :
 Drug treatment
 The animals are injected i. p. with the test drug or
saline 30 min before the unilateral 6-OHDA lesion
and 24 h thereafter.
EVALUATION
 Test sessions are performed 4, 5, 7 and 8 weeks
after 6-OHDA lesion.
 The parameters success, attempts and
sensorimotor coordination are subjected to a two-
way ANOVA with group as the independent
measure and weeks as the dependent measure.

49. TO BE CONTINUED :

50. TO BE CONTINUED :
 Scoring reaching success:-
 Reaching performance are scored by counting
misses and successful reaches for each limb.
 Scored as “reach”.
 Scored as “hit”.
Success% = no. of reaches /no. of hit x lOO

51. STEPPING TEST IN RATS
PURPOSE AND RATIONALE
o The stepping test is clinically relevant to unilateral
model of Parkinsonian akinesia.
o The 6-OHDA lesion induced marked and long-
lasting impairments in the initiation of stepping
movements with the contralateral paw which can be
ameliorated by the systemic application of drugs.

52. PROCEDURE
 6-OHDA lesion surgery
 Female Sprague Dawley rats receive two
stereotaxic injections of 6-hydroxydopamine (3.6
μg/μl in 0.2 μg/ml ascorbate-saline) into the right
ascending mesostriatal dopamine pathway using a
10 μl Hamilton syringe.

53. TO BE CONTINUED :
 Experimental setup for stepping
test
 The tests monitoring initiation time,
stepping time and step length are
performed on a wooden ramp with a
length of 1 m connected to the rat’s
home cage.
 A smooth-surfaced table is used for
measuring adjusted steps.
 During the first 3 days the rats are
handled by the experimenter to
become familiar with the
experimenter’s grip.
 During the subsequent 1–2 days the
rats are trained to run spontaneously
up the ramp to the home cage.

54. EVALUATION PARAMETER:-
 The rat is held by the
experimenter with one
hand fixing the
hindlimbs and slightly
raising the hind part
above the surface.

55. INITIATION TIME, STEPPING TIME, AND STEP
LENGTH
 Time is measured until the rat initiates movement
with the forelimb not fixed by the experimenter,
using 180 s as break-off point.
 Stepping time is measured from initiation of
movement until the rat reaches the home cage.
 Step length is calculated by dividing the length of
the ramp by the number of steps required for the rat
to run up the ramp.
 The sequence of testing is right paw testing
followed by left paw testing, repeated twice.

56. TO BE CONTINUED :
 Drug application
 Stepping tests are repeated as baseline weekly after the
6-OHDA lesion.
 The drug tests are administered during 1 day only.
Various drugs can be evaluated in weekly intervals.
 EVALUATION
 Results are expressed as means SEM.
 For statistical evaluation, the data are subjected to one-
factor analysis of variance (ANOVA) and Fisher post hoc
test.

57. BIBLIOGRAPHY :-
 Drug Discovery and Evaluation Pharmacological
Assays By H. Gerhard Vogel (Ed.) Page no. : 577-
585
 S.K Gupta, text book of screening methods and
toxicology, page no : ????/
 Tripathi KD，Essentials of medical pharmacology，
6th edition, page no:381-389.
 Rang, P.H. et al (2003) “Nuerodegenerative
Disease” Pharmacology, Elsevier Science limited,
5.497-499.
 Preclinical study of anti -parkinsonian drugs
Presented by: Sandip chaudhari

58. THANKYOU FOR YOUR ATTENTION