K.V.Archana M.pharm I

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Parkinsons disease is a degenerative disorder of the central nervous system. It results from the death of dopaminegenerating cells in the substantia nigra, a region of the midbrain and nigrostratial (dopaminergic tract); the cause of cell-death is (unknown) idiopathic. Loss of neurons in the substantia nigra pars compacta that provide dopaminergic innervation to the striatum( putamen and caudate). Replacement of dopamine could restore function.

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Function: ` Controls Voluntary Movement ` Produces the Neurotransmitter Dopamine ` Regulates Mood Location: ` The substantia nigra is located in the mesencephalon (mid brain) region of the brain. It is part of the basal ganglia.

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Dopamine has many functions in the brain, including important roles in behavior and cognition, Voluntary-movement, Motivation, Punishment and reward, Inhibition of prolactin production (involved in lactation and sexual gratification), Sleep, mood, Attention, Working memory, and learning.

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Early in the course of the disease, the most obvious symptoms are movement-related, including shaking, rigidity, slowness of movement and difficulty with walking and gait. Later, cognitive and behavioural problems may arise, with dementia commonly occurring in the advanced stages of the disease. Other symptoms include sensory, sleep and emotional problems. PD is more common in the elderly with most cases occurring after the age of 50. Resting tremor. Rigidity Bradykinesia Postural instability. Facial mask. Anosmia .

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1. Genetic causes ( alpha synuclein and park gene). 2. Environmental causes. -MPTP. -ROTENONE.

3. Welders exposure. (manganese from the fumes of welding rods).

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The pathology of the disease is characterized by the accumulation of a protein called alphasynuclein into inclusions called Lewy bodies in neurons, and from insufficient formation and activity of dopamine produced in certain neurons within parts of the midbrain.

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Early signs and symptoms. DaT scan( uses a drug, injected into the blood stream to assess the dopamine containing neurons by analyzing the images then taken by a gamma camera). PET imaging scan may show low levels of dopamine in the brain - a key sign of the disease.

I Drugs affecting brain dopaminergic system: x Dopamine precursor: Levodopa ( l-dopa). x Peripheral decarboxylase inhibitors: Carbidopa , Benserazide. x Dopaminergic agonists : Bromocriptine , Ropinirole , Pramipexole. x MAO-B inhibitor: Selegiline. x COMT inhibitor: Entacapone , Tolcapone. x Dopamine facilitator : Amantidine. II Drugs affecting brain cholinergic system : ( a) Central anticholinergics: Trihexyphenidyl( benzhexol) , Procyclidine , biperidin. (b) Anti-histaminincs: Orphenadrine , Promethazine.

The various methods used for the screening of anti-parkinsons disease are categorized into two: IN VIVO METHODS: ` Tremorine and oxotremorine antagonism. ` MPTP models in monkeys. ` Reserpine antagonism. ` Elevated body swing test. ` Skilled paw reaching in rats. ` Stepping tests in rats. IN VITRO METHODS: ` Experiments using rat striatal slices. ` Dopamine stimulated adenyl cyclase activity. ` Radio ligand binding studies for D1 and D2 Dopamine receptors. ` Dopamine release from synaptosomes.

1.Tremorine and oxotremorine antagonism. Purpose and rationale: The muscarnic agonists tremorine and oxotremorine induce parkinsonism like signs such as tremor, akinesia, spasticity, salivation, lacrimation and hypothermia.These signs are antagonized by anticholinergic drugs.

Groups of 6-10 male NMRI mice weighing 18-22 g are used

They are dosed orally with the test compound or the standard(5mg/kg benzatropine mesilate) 1hr prior to administration of 0.5mg/ kg oxotremorine injection.

Rectal temperature is measured before administration of the compound and 1,2,3, hours after oxotremorine injection.

Tremor is scored after oxotremorine dosage in 10 seconds. observation period is maintained as 15 mins for 1 hour.

In a similar manner salivation and lacrimation are scored 15 and 30 minutes after oxotremorine injection.

TREMOR Absent Slight Medium Severe

SCORE 0 1 2 3

Evaluation: Hypothermia: The difference of body temperature after 1, 2 and 4 hours 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 three observation periods (1, 2 nd hours) are summarized. The numbers in the treated groups are expressed as percentage of the control groups. Salivation and lacrimation: The scores for both the symptoms for all in each group at the 3 observation periods ( 1, 2 and 4 hours).The number in the treated groups are expressed as percentage of the control group.

2. 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 Parkinsons disease in exposed individuals. When administered to primates this compound causes a partial destruction of basal ganglia and a syndrome that resembles Parkinsons disease

8 adult rhesus monkeys weighing 5-8 kg over a period of 5-8 days are injected with cumulative iv doses of N-methyl-4-phenyl -1,2,3,6 tetrahydropyridine(NMPTP) upto 10-18 mg/kg.

These animals showed a parkinsonism like disorder (akinesia, rigidity, postural tremor, flexed posture, eyelid closure, drooling) which was reversed by the administration of L-dopa.

The pathological and biochemical changes produced by N-MPTP are similar to the well established changes in patients with Parkinsonism.

The N-MPTP intoxication was applied using marmosets to evaluate potential antiparkinsonian drugs.

Evaluation: The severity of parkinsonian symptoms are rated by trained observers using a scale of 0 (normal) to 17 (maximum severity) that assesses. Movement (0-normal, 1-reduced, 2-sleepy). Checking movements (0-present, 1-reduced, 2-absent). Attention and blinking (0-normal, 1-abnormal). Posture (0-normal, 1-abnormal trunk, 2-abnormal trunk and tail, 3abnormal trunk, tail and limbs, 4-flexed posture). Balance and coordination (0-normal, 1-impaired, 2-unstable, 4-falls). Reactions (0-normal, 1-reduced, 2-slow, 3-absent). Vocalizations (0-normal,1-reduced,2-absent).

3. 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. This phenomenon can be antagonized by dopamine agonist.

Evaluation: Loco motor activity and grooming scores of drug treated animals are compared with control.

Male NMRI mice weighi ng 2025 gm are used

They are injected (i.p.) 5mg/kg reserpin e and tested 24 hours

Drugs are administ -ered 30mins prior to the observat -ion.

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Animals are placed singly on the floor of a perspex container situated on a pan lab proximity tester

Horizontal movement s are recorded for 10 mins

Moreover rearings and grooming episodes are registered by an experienced observer.

In vitro Experiments using rat striatal slices: Purpose and rationale: Striatum is the brain region , which is primarily effected in parkinsonism. The release of the neurotransmitters like dopamine and ACH in response to test agent serves as a good in vitro marker of its activity.

Male Sprague Dawley rats (150250 gms) are cervically dislocated; the skull is opened and the right and left striata are removed and placed in ice cold Krebs solution

The striata is cut into 0.4mm thick slices using a tissue chopper . the slices are kept floating for 30 minutes inn krebs solution continually gassed .

Labeled slices are transferred to super fusion chambers and perfused with krebs solution at 37c at a flow rate of 0.5ml/min. After washing and stabilization , 5 min fractions of super fusate are collected.

The perfusion buffer contains 1mM hemicholinium to inhibit choline uptake.

The slices are subjected to field stimulation with rectangular pulses of alternating polarity, with a current strength of 10-15 mA/cm and a pulse duration of 2 sec at a stimulation frequency of 3Hz for 5 mins

Drugs to be tested are present in the super fusion fluid. The radioactivity in the superfusate samples and in the tissue is determined by liquid scintillation counting.

Radio ligand binding studies for D1 and D2 dopamine receptors. Male Sprague Dawley rats(150-200 gms) are cervically dislocated and the right and left striata removed.straiatal tissue is homogenized with Teflon glass homogenizer in 20 volumes of ice cold buffer containing 50 mM tris-Hcl, pH7.4, 2 mM EGTA, and 10 % sucrose.

The homogenate is centrifuged for 5 min at 800 gms, and the supernatant is centrifuged for 20 mins at 49,000 gms. The pellet is washed and suspended in 50 mM Tris- Hcl buffer, pH 7.4. Reaction mixtures containing 50 mM Tris-Hcl buffer, pH 7.4, 120mM NaCl , 5 mM KCl , 2mM CaCl2, 1mM MgCl2 and [3H] SCH 23390 (0.1-6.4nM) or [3H] Raclopride (0.2516nM) in a total volume of 250l is incubated at 37 0C for 30 minutes.

And terminated by vaccum filtration through whatmann filter. Further, washing with cold 50 mM Tris-Hcl buffer, pH 7.4, may be given.

Nonspecific binding is defined as binding in the presence of 10M cisflupenthixol for D1 dopamine receptor binding or 1 M haloperidol for D2 dopamine receptor binding.

The receptors densities and affinities are calculated by scathard analysis.

Dopamine-stimulated Adenylyl cyclase activity. Male Sprague dawley rats (150-250 gms) are decapitated and the right and left striata are removed.

Striatal tissue is homogenized by Teflon glass homogenizer in chilled buffer containing 10mM imidazole, 2mM EGTA and 10% sucrose, pH 7.3 .

The homogenate is centrifuged at 1000 g for 10 minutes and the supernatant that is obtained is re-centrifuged at 27,000 g for 20 min. The pellet obtained is washed twice and suspended in 10mM imidazole, pH for 20 min.

The pellet obtained is washed twice and suspended in 10mM imidazole, pH 7.3.

The membrane protein is determined by Bradfords method using bovine serum albumin as standard.

Adenylyl cyclase activity is measured by calculating the conversion rate of [32P] ATP to [32P] cAMP. The assay is performed in 250l of solution containing 10mM imidazole , pH 7.3, 2mM MgCl2, 0.1 mM papaverine,0.2mM EGTA,1 mM dithiothreitol,1M GTP, 0.1mM ATP, 2Mm phosphocreatine, 5 units of creatine phospho kinase , and 1Ci of [ -32P] ATP.

The reaction mixture is preincubated at 30oC for 5 min , and the reaction is initiated by adding 50 to 60 g of membrane proteins and incubated for an additional 10 min.

The reaction is terminated by the addition of 300l of stopping solution(2% SDS, 25mM ATP, and 1.3 mM cAMP).

Formed [32P] cAMP is separated from [32P] ATP by chromatography.

The greatness of a nation and its moral progress can be

judged by the way its animals are treated- MAHATMA GANDHI.

Etiology of parkinsonism.-5M (3 times) MPTP model of screening anti parkinsonism drugs. -5M (4 times) Methods of screening of anti Parkinsonism drugs. -10M Pathophysiology of parkinsonism.-5M