preclinical ways of developing a new drug for treatment of Parkinson's disease

1. Evaluation of Drug for
Parkinson’s Disease
Dr. Shakeeb Dhorajiwala

2. Outline
Conclusion
Clinical Evaluation
Drug Evaluation In-vivo Behavioural models
Drug Evaluation In-vitro models
Therapeutics
Clinical presentation
Etiology
Introduction
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3. Introduction
• Parkinson’s disease (PD): Neurological syndrome characterized by:
• bradykinesia,
• postural instability,
• rigidity and
• involuntary tremors.
• Biochemistry:- depleted dopamine (DA= 50-70% DAergic neuron loss) and
relatively increased acetylcholine (Ach) in affected area.
• Enormous disease burden with incidence rates (IR) from 1.5-20/ 1
lakh/year.1
• mean age of presentation > 55 years.
• Pathognomic histopathology: Lewy bodies (α synuclein and ubiquitin)
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1.Das SK. Et al. Epidemiology of Parkinson’s disease in the city of Kolkata, India: A
community based study . Neurology 2010;75(15):1362-9

4. Historical Aspect
Parkinson's disease (PD) was first
recognized by James Parkinson in
1817
• Wrote an essay on shaking palsy
The term PD coined by “Jean
Martin Charcot”
Swedish scientist Arvid Carlsson in
1950 identified underlying
biochemical changes in the brain
• Awarded the Nobel prize
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5. Etiology
i. Idiopathic
ii.
Arteriosclerotic
iii. Post-
encephalitic
iv. Hepato-
lenticular
degeneration
v. Drug induced
(haloperidol/
chlorpromazine)
vi. Oxidative
stress
vii. Toxins like
rotenone/permeth
rin
viii. Heavy
metals like iron
and manganese
ix. Protein
aggregation &
phosphorylation
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6. Parts of Brain Affected and Its Effects
• Components of Brain involved:
i. Corpus striatum (caudate nucleus and putamen)
ii. Globus Pallidus
iii. Substantia Niagra- Pars compacta
• Part affected and its effects:
a) Depletion of DA neuron postural instability and rigidity
b) Degeneration of norepinephrine secreting locus ceruleus autonomic
symptoms and depression
c) Degeneration of cholinergic nucleus basalis dementia
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7. Clinical presentation
Four cardinal motor symptoms:
i. Bradykinesia
ii. Muscular rigidity
iii. Resting tremor
iv. impairment of postural
balance leading to disturbances
of gait.
Other symptoms: Gait and
posture disturbance Stooped
forward – flexed posture,
Festination etc.
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8. Speech, Facial and Swallowing Disturbances
a) Hypophonia
b) Festinating speech
c) cognitive disturbance
d) Dysphagia
e) Masked facial expression
f) oculogyric crisis
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9. Diagnosis
1. Medical history and neurological examination
2. The unified parkinson’s disease rating scale (UPDRS)
3. Autopsy – 75% of confirmed diagnosis of PD
4. MRI/CT scan – May appear normal
5. 18F PET scan – Decreased dopamine activity
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10. Pharmacotherapy (1/2)
Dopaminergic
system:
Dopamine
precursor
Levodopa
Dopaminergic
agonist
Bromocriptine
Ropinirole
Pramipexole
COMT-
inhibitors
Tolcapone
Entacapone
Glutamate
(NMDA receptor)
agonist
Amantadine
Peripheral
decarboxylase
inhibitor
Carbidopa
Benserazide
MAO-B
inhibitors
Selegiline
Rasagiline
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11. Cholinergic
system
Central
anticholinergics
Trihexyphenidyl Procyclidine Biperiden
Anti-
histaminics
Orphenadrine Promethazine
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12. Drug Evaluation:
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13. 10-09-2020 14
• In vitro primary microglial culture
• Experiments using rat striatal slices
• Dopamine stimulated Adenylyl
Cyclase activity
• In Vitro neuroprotective efficacy
In-vitro
Models

14. In-vitro Primary Microglial Culture
Purpose
• Microglia are mainly affected in Parkinson
disease.
• Culture of primary microglia are useful for
assessing inflammation and
neurodegenerative processes in vitro.
Procedure..
• Primary rat microglial enriched cultures
obtained from cerebral cortices of neonatal
rat.
• Cortices sectioned & washed in Hank’s
Balanced Salt Solution.
• Cell suspension obtained is diluted in medium
containing 10% horse serum and centrifuged.
• Cells cultured in Minimum Essential Eagle
supplemented with 10% horse serum,
glucose, Glutamax, Penicillin, Streptomycin
and GM-CSF and maintained in flasks at 370C.
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15. Procedure contd..
• Assay performed in solution
containing imidazole,
papaverine, Mgcl2, GTP, ATP,
phosphocreatine, creatine
phosphokinase, [32P] ATP
• Reaction mixture preincubated
for 5 min & reaction initiated
by adding membrane protein.
• Reaction terminated by adding
stopping solution[ATP,cAMP]
Assessment
• Adenylyl cyclase activity
measured by calculating
conversion rate of [32P]ATP to
[32P]cAMP.
• Formed [32P] cAMP separated
from [32P] ATP by
chromatography.
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In-vitro Primary Microglial Culture

16. Experiments Using Rat Striatal Slices
Purpose & Rationale
• Striatum – Primarily affected in
parkinsonism
• Release of neurotransmitters like DA &
Ach  good in vitro marker of its
activity
Procedure..
• Male Sprague Dawley rats decapitated
& rt. and lt. striata removed.
• Placed in Krebs solutions
• Striata cut into thick slices and kept
floating for 30 min in Krebs solution
continuously gassed with 95% O2 &
5% CO2 at room temp.
• Slices labeled by incubating for 30 min
with [3H] dopamine and [14C] choline.
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17. Experiments Using Rat Striatal Slices
Procedure contd..
• Labeled slices transferred to super fusion chamber and perfused with
Krebs solution.
• After washing & stabilization, fractions of superfusate are collected. The
perfusion buffer contains 1mM nomifensine to inhibit dopamine
reuptake and 10mM of hemicholinium to inhibit choline uptake.
• Drugs to be tested are present in perfusion fluid
Assessment
• radioactivity in superfusate samples and in tissue is determined by liquid
scintillation method
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18. Dopamine stimulated Adenylyl Cyclase
activity
Purpose and rationale
• Dopamine levels are decreased in
Parkinson’s patients.
• Dopamine increases cAMP formation by
stimulating adenylyl cyclase activity.
Procedure..
• Male Sprague dawley rats decapitated
and striata removed.
• Striatal tissue homogenized by Teflon
glass homogenizer in chilled buffer.
• Homogenate centrifuged for 10 min &
supernatant recentrifuged for 20 min.
• The pellet obtained washed &
suspended in imidazole.
• Membrane protein determined by
Bradford’s method using bovine serum
albumin as omparator.
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19. Dopamine stimulated Adenylyl Cyclase activity
Procedure contd..
• Assay is performed in solution
containing imidazole, papaverine,
Mgcl2, GTP, ATP,
phosphocreatine, creatine
phosphokinase, [32P] ATP
• Reaction mixture preincubated
for 5 min & reaction initiated by
adding membrane protein.
• Reaction terminated by adding
stopping solution[ATP,cAMP].
Assessment
• Adenylyl cyclase activity
measured by calculating
conversion rate of [32P]ATP to
[32P]cAMP.
• Formed [32P] cAMP separated
from [32P] ATP by
chromatography.
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20. In Vitro neuroprotective efficacy
Purpose & Rationale
• Oxidative stress causes loss of
dopaminergic neurons in Substantia
nigra.
• Neuroprotection to avoid neuronal
death been proposed as future therapy
for neurodegenerative disorders.
• Viability of cells using suitable test in
presence oxidants helpful in finding
neuroprotective agent.
Procedure..
• Human neuroblastoma cells, SH-SY5Y
cultured and maintained in Dulbecco’s
modified Eagle’s medium containing
10% fetal calf serum and 1%
penicillin/streptomycin antibiotic
mixture.
• Cells grown in 95% air and 5% CO2 at
370 for 24 h.
• SH-SY5Y cells are seeded at a density of
4 x 104 cells/well in 96 well culture
plates.
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21. In Vitro neuroprotective efficacy
Procedure contd..
• Cells subjected to stress by incubating with
or without H2O2.
• Appropriate concentration of test agent
added to culture plate 0.5 h before H2O2 to
evaluate its efficacy as neuroprotective
agent.
Assessment
• Cell survival evaluated by performing MTT
assay.
• MTT added to cultures at 0.02mg/ml and
after incubation.
• Media removed carefully and reaction is
stopped by adding isopropanol containing
0.04 N HCL.
• Absorbance of each well measured using
microplate reader.
• Increase in viability of cells in test walls
(Develop purple color) indicate efficacy of
the test drug as neuroprotective agent
against an oxidizing agent.
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22. • Reserpine antagonism
• Neuroleptic induced parkinsonism
• MPTP model
• Surgical Induction
• Cholinomimetic induced Parkinsonism
• Elevated swing test
• Skilled paw reaching test
• Stepping test
In-vivo
models:
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23. Reserpine antagonism
Reverses the akinetic state
Administration of dopamine agonists
Akinetic state
Depletion of catecholamines
Systemic administration of reserpine
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Percentage inhibition of peak reserpine effect is
evaluated
Test/standard control is administered(After 30
mins of reserpine inj)
Hind limb rigidity, arched body position, fixed facial
expression, ptosis
Markedly hypokinetic movements (after 20-30 mins
of inj)
Either IV(5 mg/kg) or IP(2.5 mg/kg) of
reserpine
NMRI Mice(either sex)
Purpose & Rationale Procedure

24. I)Reserpine Antagonism:
Assessment of
hypokinesia
wooden box of 88cmx 88cm
x60 cm with floor divided into
16 equal squares.
No. of squares entered /2 min
by rat counted for total
duration of 120 min.
hypokinetic rat score
reduced significantly.
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25. II) Assessment of
muscular rigidity
Simple grasping test assess
muscular rigidity of animal
metal rod held at height 50
cm above table.
Rat made to grasp rod with
forelimb total time for
which it remains on bar is
noted.
Scores of drug treated
animals compared with
controls by ANOVA.
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26. III) Assessment of
Catatonia:
Catatonia: most obvious behavioural
response to DA antagonist.
Its due to dysfunction at cortico-sub-
corticol level.
A grading scale used to compare effects
of drugs.
Symptoms strongly simulate that in
human.
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27. Reserpine antagonism
Advantage
• Help to stimulate
symptoms of
parkinsonism effectively.
Disadvantage
• Can not be used to screen
potential neuroprotective
agents as reserpine
causes depletion of
biogenic amines and not
neurodegeneration.
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28. Neuroleptic Induced Parkinsonism
Purpose & Rationale
• Phenothiazine derivatives-
trifluperazine, chlorpromazine
produce typical EPS by blocking
striatal dopamine receptors.
• Used as pharmacological tool to
induce parkinsonism in lab animals.
Reversal of neuroleptic effect is quantitated
with behaviour scoring system
Hypokinesia + tremors + rigidity
On chronic administration (Chlorpromazine 2
mg/kg ip for 7 days)
muscular rigidity + bradykinesia + catatonia
Administration of single dose 4 mg/kg ip
chlorpromazine in rats
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Procedure

29. Assessment of
tremors:
• Link for tremors:
https://www.youtube.com/watch?
v=bcmcjLuB_bs
Test agents with anti-parkinson effect
significantly reduces scores.
Numbers in treated groups are expressed as
% of number of control group.
Done for every 15 min for 1 hr for each
group (Vehicle, standard, Test)
Forelimbs maintained in this position for 10 sec and
observed for body/hind limb tremors and scored
on degree of tremors on an arbitrary scale.
Rat lifted by tail and suspended approx. 8 cm
above surface.
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Degree of tremor score
Absent 0
Slight 1
Medium 2
Severe 3

30. Neuroleptic Induced Parkinsonism
Advantage
• Mainly used to evaluate
anticholinergic drugs  effective in
ameliorating the sign and
symptoms of neuroleptic induced
parkinsonism in rodents.
Disadvantage
• Catatonia not induced.
• L-Dopa- DOC to control tremors
shows partial effect.
• Anti-cholinergic with less efficacy
in humans shows remarkable
response in this model.
• Blocks DAergic neurotransmission
& not neurodegeneration.
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31. MPTP Model
Partial destruction of basal ganglia and
syndrome that resemble PD.
Lipid peroxidation of striatal membrane
Reactive free O-
↑ extracellular DA levels in striatum
1methyl,4phenylpyridine(MPP+) by MAO-B in
brain.
1 methyl, 4 phenyl, 1,2,3,6 tetrahydropyridine
(MPTP)
After 6-8 weeks, syndrome stabilizes and experiment
to test antiparkinson agents can be initiated.
The animal become akinetic f/b stooped posture &
aphagia
Exhibit syndrome immediately after injection, characterized
by agitation, ataxia, myoclonus, lingural dyskinesia for 10-30
min
MPTP 0.3 mg/kg IV/SC daily for 5-8 days
Cynomolgus monkeys - 2-4kg
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Purpose & Rationale Procedure

32. MPTP Model
Assessment
•Tremors & rigidity EMG
•Akinesia Photo-cells
•The severity of Parkinson symptoms is rated by
trained observer using scale of 0 (normal) to 17
(max. Severity)
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33. MPTP Model
Advantage
• Best animal model of PD
primarily in primates and later
in mice
• Produces same syndrome as in
clinical condition
• Serve as excellent platform to
test new drug before initiating
them for patients
Disadvantage
• Primate model is expensive
and tedious to carry out.
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34. Surgical Induction
Direct lesion of nigrostriatal DAergic neurons by injecting 6-OH-
DA/ electrolesion are selective and reproducible ways to
simulate clinical conditions in animals.
After surgical induction of neurodegeneration two activities
are recorded:
• Spontaneous motor activity
• Rotational behaviour
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35. Spontaneous Motor Activity
Purpose and rationale
• Neurotoxin 6-OHDA induces
permanent & selective damage of
neuronal region where it is injected.
• Altered level of monoamines NE,
5HT & DA induce behavioral deficits
• In rats, inhibition of spontaneous
locomotor activity, hypokinesia,
aphagia, increase in pain sensitivity
– typical effects
A slow infusion of respective agents.
Prefilled probe drive with microliter syringe mounted
on the top.
Burr hole made at point of appropriate stereotaxic
coordinates
Longitudinal midline incision with skin retraction.
Rats placed on stereotaxic apparatus
Anesthetized with ketamine hydrochloride 80 mg/kg
Adult male rats weighing 200-250 gm
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Procedure..

36. Spontaneous Motor Activity
Animal is allowed to recuperate for 10-15 days
before the experiments
Wound sutured
After 5 min of injection, infusion probe withdrawn
Ascorbic acid – To prevent auto-oxidation.
Test group administered with 8 µg 6-OHDA & control group
administered with equivalent amount of vehicle control.
Interruptions are significantly reduced in hypokinetic
rat.
Total number of interruptions of photocell beam for
a predecided duration recorded.
Rat introduced & allowed to acclimatize for 10-15
min.
Fitted with photocell beam
Well lit sound dimmed chamber with a transparent
acrylic cage.
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AssessmentProcedure contd..

37. Rotational Behavior in Rats
U/L lesion of DAergic nigrostriatal pathway in rat by
neurotoxin 6-OHDA induces selective presynaptic terminal
loss of DA neurons with hypersensitivity of postsynaptic
dopaminergic receptors on lesioned side.
I/L side  Indirect acting compound e. g.
Amphetamine
C/L side Directly acting dopamine agonists e.
g. Apomorphine
Test is used for study of central dopamine
function and evaluation of DA antagonists and
agonists. Animal allowed to recuperate for several weeks
and for development of lesion.
Total of 8 µg 6-OHDA in NS injected and wound
sutured.
Prefilled probe drive with microliter syringe is
mounted.
Sagittal section made and 2 mm wide burr-holes
drilled.
Rats placed on stereotaxic apparatus
Anesthetized with sodium phenobarbital
Male Wistar rats 200-250 gm
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Purpose & Rationale Procedure

38. Rotational Behavior in Rats
• Specially opaque plastic sphere
attached to solid stage
programming equivalent- test
chamber.
• No. of full turns either I/L or C/L to
the lesion, is recorded as an
automatic printout every 15 min
for 1-2 hr test sessions.
• To determine control values for
ipsilateral turning, each rat is
administered with 2.5 mg/kg of
amphetamine and placed in
chamber.
• Similarly, to determine control
values for C/L turning, each rat
administered with 1 mg/kg of
apomorphine and placed in
chamber.
• Test compounds are given i.p./s.c.
and animals are placed into circling
chambers for 2 hr.
• % change of drug turns from
control turns is recorded.
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Assessment contd..Assessment

39. Rotational Behavior in Rats
Advantage
• Rotational response to
amphetamine administration
is widely used as behavioral
index to evaluate success of
the 6-OHDA lesion in the
rodent model of PD, and it
has been shown to correlate
with the extent of the lesion.
Disadvantage
• Amphetamine-induced
rotation test is a poor
predictor of development of
dyskinesia
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40. Clinical Evaluation:
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41. Challenges in conducting studies
• Clinical studies in PD are hampered by:
• Long duration and slow progressive course of disease.
• Variability and heterogeneity of symptoms and signs,
• Cyclic episodes in severity of the symptoms/signs over day related to the time
of medication and polytherapy.
• In addition, misdiagnosis, comorbidity and co-medication add to the
heterogeneity of the patient population.
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42. Selection Criteria
Inclusion Criteria:
• Patients presenting classical
features of PD
• Patients with positive L-Dopa test
in early PD
• Participant is willing and able to
give informed consent and willing
to commit long term follow up.
Exclusion Criteria:
• H/o hepatitis B & C, CMV /drug or alcohol
induced hepatic toxicity.
• Atypical parkinsonism, cognitive impairment,
bipolar disorder, schizophrenia and other
psychiatric disorders.
• H/o repeated strokes or head injuries or a
stroke within 3 months.
• H/o any clinically significant cardiovascular
events or procedure for 6 months, including
myocardial infarction, angioplasty, unstable
angina or heart failure.
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43. Trial Objectives
• The design of the clinical trials in PD depends on the objectives of the
study.
1. Symptomatic relief in early stage PD before L-Dopa+ treatment;
2. Symptomatic relief in patients with PD on L- Dopa+ subdivided in:
i. Patients on L-Dopa+ with insufficient control of motor symptoms;
ii. Patients on L-Dopa+ with motor fluctuations;
iii. Patients with serious unpredictable and rapid changing motor
fluctuations.
3. Therapies aimed to modify disease progression
i. To postpone late motor fluctuations
ii. To delay disease progression
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44. Phases of Clinical Trial of Anti-
Parkinson’s Drug:
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45. Phase I
• Objectives:
• Assessment of safety, tolerability, PK
and PD
• Pharmacokinetic study:
• Pharmacokinetic study of drug
thoroughly determined with respect
to absorption, bioavailability and
route of elimination.
• Pharmacodynamic study:
• No specific human pharamcodynamic
models.
• Safety assessment:
• Identified adverse effects be
characterized in relation to duration
of treatment, dosage, recovery time
and age.
• Clinical observations be
supplemented by appropriate
laboratory tests.
• Assess adverse effects
• Neurological
• Psychiatric
• Endocrinological
• Cardiovascular
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46. Phase I
• Study group: small no. of healthy volunteers (20-80)
• Dosing:
• Dose escalation study to determine therapeutic dose.
• Titration of dose of new drug until optimal effect /maximal tolerated dose
reached.
• In maintenance period, patients should stay at their individual determined
optimal dose level.
• Criteria for an optimal effect and intolerance should be defined in the study
protocol.
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47. Phase II
• Phase IIa
• Initial therapeutic studies
• Purpose : to obtain initial
information on safety/ establish
preliminary evidence of
activity/suitable therapeutic dose
ranges and frequency of dosing.
• Dose ranging studies in controlled
titration, using at least 3 dosages,
to establish lower clinically
effective dose range & optimal
dose.
• Phase IIb
• Efficacy endpoints depend upon
study objective
• For assessment of motor function
in PD, the UPDRS(Daily activity)
and UPDRS (Motor examination)
are accepted and validated scales.
• OFF time or ON time is the main
efficacy variable, an operational
definition of OFF and ON period
should be established.
• Study size: 50 - 300
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48. Phase III
• Objectives:
• To confirm efficacy and safety.
• Drug interactions
• Efficacy is assessed on the basis
of UPDRS rating in patients with
PD.
• Safety assessment is done by
monitoring adverse effects.
• Drug interactions:
• All pk and pd interactions between
test drug and antiparkinson drugs
should be studied.
• Also potential pd interaction with
alcohol and CNS active drugs
should be investigated.
• Study group: Large number of
patients 250 - 1000
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49. Phase IV
• Objectives
• Post marketing surveillance
• Detect rare adverse effects, drug interaction
• Long term safety
• Effect on mortality and morbidity
• Quality of life
• Study group: Large patient population taking the drug
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50. Conclusion
• Better understanding of nature, time course and molecular players in
genesis will help to elucidate the mechanisms of cell death in
Parkinson’s disease.
• Addressing these lacunae may prove useful strategy for the
development of future preventive and therapeutic agents for this
neurodegenerative disorder.
• Appropriately designed models, which allow such investigations are
essential.
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51. ou
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