Antiparkinsonian Activity in animals. screening methods

anti - parkinsonian activity

1. A NTI – PARKINSONIAN ACTIVTY Dr.S.Santhosh Post - Graduate

2. Specific Learning Objectives Introduction to Parkinson’s disease Clinical features Drugs causing Parkinson – like symptoms Various screening techniques for evaluation of anti – Parkinsonian drugs

3. Introduction Affects basal ganglia Neurochemical origin discovered by Hornykiewicz 1960 Dopamine content in substantia nigra and corpus striatum in post mortem brain was extremely low Later correlated to almost complete loss of dopaminergic neurons from substantia nigra and degeneration of nerve terminals in the striatum

4. Symptoms : Hypokinesia – Dopamine Rigidity, Tremors – Ach, 5HT, NE, GABA, DA Experimental lesion : secondary consequences - hyperactivity of remaining dopaminergic neurons – increased rate of transmitter turnover - increase in the number of dopamine receptors – state of denervation hypersensitivity Important for the therapeutic effectiveness of levodopa

5. Tremorine and Oxotremorine Tremors These centrally as well as peripherally acting Cholinergic agents known to induce Parkinsonism like signs and symptoms in rodents – tremors, ataxia, spasticity, lacrimation, salivation, hypothermia, etc Prevented by centrally acting anticholinergic drugs – used in treatment of parkinsonism for long time

6. Methodology : Groups of 10 male swiss mice – 20-25gm Treated with test compound or standard orally 1hr prior to administration of 0.5mg/kg Oxotremorine s.c Rectal temp measured before and 1, 2, and 3 hr after Oxotremorine injection Tremors are scored in 10sec observation periods every 15min for 1hr Salivation and lacrimation are scored 15 and 30min after Oxotremorine injection

7. Grade : Tremors, Salivation, Lacrimation -- score Absent 0 Slight 1 Moderate 2 Severe 3 Average values are statistically compared between test/standard and control groups. Reliable method for testing centrally acting anticholinergic drugs like Benztropine

8. Watanabe et al (1997) – rats treated with 3-acetylpyridine show a selective degeneration of neurons in the inferior olive nucleus and the olivo -cerebellar tract with characteristic motor incoordination and ataxia To measure the effect of 3-acetylpyridine, the maximum height of vertical jump stimulated by foot shock is measured

9. MPTP Induced Parkinsonism in Monkeys N-methyl -4- phenyl-1,2,3,6-tetrahydropyridine Irreversible destruction of nigrostriatal dopaminergic neurons Acts by converting MPTP to MPP+ by MAO-B MPP+ taken up by dopamine transport system and thus acts selectively on the dopaminergic neurons – inhibit mitochondrial oxidation reaction producing oxidative stress.

10. Methodology : 8-10 adult rheus monkey 6-8kg Treated over a period of 5-8 days with cumulative i.v doses of 10-18mg/kg of MPTP Exhibits typical parkinsonism like disorder Symptoms are reversed by treatment with Levodopa Pathological and biochemical changes produced by MPTP is similar to well established changes seen in parkinsonism patients

11. The severity of Parkinsonism induced MPTP in monkey is scored and graded using a scale of 0 (normal) and 17 (maximum severity)

12. Criteria Score Score 1 Score 2 Score 3 Score 4 Movement Normal Reduced Sleepy ------------ ---------------- Checking movements Present Reduced Absent -------------- ---------------- Attention and blinking Normal Abnormal -------------- -------------- ----------------- Posture Normal Abnormal trunk Abnormal trunk and tail Abnormal trunk , tail and limbs Flexed posture Balance and co ordination Normal Impaired Unstable Falls ----------------- Reaction Normal Reduced slow Absent ----------------- Vocalization Normal Reduced Absent -------------- -----------------

13. Reserpine Antagonism Reserpine – known to produce significant depletion of central catecholamine stores and biogenic amines like 5 HT Produce marked sedation in mice shortly after administration which is followed by ptosis, hypokinesia , rigidity, catatonia and immobility Antagonised by Dopamine agonists which is used in treatment of Parkinsonian

14. Methodology : swiss mice of either sex 20-30gms Treated with 5mg/kg i.p 24hrs before test Test and standard drug is administered 30 min before recording the observation Animals are placed in individual Perspex cages (30x26x20cm) Horizontal movements are recorded for 10min Other effects like rearing and grooming episodes are recorded

15. Locomotor activity and grooming scores of control and treated animals are subjected to ANOVA test for assessing the significance of observations Develop spontaneous orofacial dyskinesia similar to tardive dyskinesia observed in humans Incidence of tongue protrusion quantify the severity of oral dyskinesis

16. 6-hydroxydopamine induced Nigrostriatal lesions in Rats 6-HDA neurotoxin – U/L dopaminergic nigrostriatal pathway lesion Hypersensitivity of post synaptic dopaminergic receptors in the striatum of lesioned side Rats rotate towards - lesioned side (I/L) when indirectly acting compound like Amphetamine is given - opposite side (C/L) when directly acting agonist like Apomorphine or the dopamine precursors Levodopa is given

17. Used in the evaluation of the dopamine agonist and antagonists Methodology : Male wistar rats 200-250gm Housed individually in a controlled environment with free access to food and water Anaesthetised with Pentobarbitone sodium Head placed in a stereotaxic device Positioned according to Atlas of Konig and klippel

18. Surgical cut in the skull – 2mm hole drilled (avoid injury to meninges) 30 gauge stainless steel cannula connected to Hamilton syringe placed at anterior Zona compacta of substantia nigra 8 μ g of 6-HDA injected intra-cranially wound closed animal allowed several weeks for recovery and development of Nigrostriatal lesion

19. Test chamber – opaque plastic sphere attached to the solid state programming equipment No of full turns I/L or C/L side to the lesion – recorded on automatic print out counter every 15 min for 2 hr test session The test drug and standard are given i.p or s.c After 30 mins For determining the control values - I/L turning - 2.5mg/kg of d-amphetamine s.c - C/L turning – apomorphine 1mg/kg s.c

20. the circling movements are recorded for 2 hr Percentage of change of drug turns from control turns is recorded and the ED50 values are calculated

21. Elevated body swing test Borlongan and Sanberg 1995 Measure of assymetrical motor behaviour of hemiparkinsonian animals in drug free state in rats lesioned with 6-HDA

22. Skilled paw reaching test Barneout et al 1996 U/L inj of 6-HDA into medial forebrain bundle Producing impairment of paw reaching on both sides Can be ameliorated by drug treatment Test not well established for routine screening of Antiparkinsonian drugs

23. Thank you