Screening of antiparkinson agent

Selection of models during study of antiparkinson agents

1. SCREENING OF ANTIPARKINSON AGENT PREPARED BY: SONAL VIJAY PANDE MPHARM: SEM 1 DEPARTMENT :PHARMACOLOGY

2. Introduction Etiology Pathophysiology Classification Mechanism of action In vivo preclinical evaluation In vitro preclinical evaluation OVERVIEW

3. INTRODUCTION 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. Features Hypokinetic movement Akinetic- Difficulty in initiating movements & decreased spontaneous movements. Bradykinesia – Slowness of movement. Decreased associated movements. 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.

4. ETIOLOGY

5. MPTP IT is byproduct of the illicit manufacture of synthetic meperidine derivative MPTP MPPC (1,2,3,6-methyl –phenyl- MAO-B ( pyridinium ion) Tertahydopyridine ) MPPC is taken up by dopaminergic neurons and cause mitochondrial complex I (ERC) defect similar to that found in PD.

6. Alpha- synuclein Abnormal accumulation of this protein in brain cells cause damage by binding with ubiquitin in the damaged cells. This protein accumulation forms proteinaceous cytoplasmic inclusions called lewy bodies. Latest research shows that death of dopaminergic neurons by alpha-synuclein is due to defect in machinery that transports proteins between two major organelles-the endoplasmic reticulum (ER) and Golgi apparatus.

7. OXIDATIVE STRESS Because of oxidative metabolism of dopamine to yield hydrogen peroxide H2O2 and other reactive oxygen species (ROS). Oxidative stress and consequesnt cell death could develop in SNc under such circumstances in which there is : a) increase dopamine turnover ,resulting in excess peroxide formation . b)A deficiency in glutathione thereby diminishing brain capacity to clear H 2 O 2 . c)increase in reactive iron which can promote OH radical formation .

8. PATHOPHYSIOLOGY

9. CLASSIFICATION 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. Antihistaminics : Ophenadrine , Promethazin .

10. SCREENING MODELS INVIVO INVITRO

11. SCREENING MODELS INVIVO METHODS Tremorineand OxotremorineAntagonism MPTP Model of Parkinson’s Disease Reserpine Antagonism Circling Behavior in Nigrostriatal Lesioned Rats Elevated Body Swing Test Skilled Paw Reaching in Rats Stepping Test in Rats Gait analysis Rotenone induced parkinsonism TransgenicAnimal Models of Parkinson’s Disease Cell Transplantationsinto Lesioned Animals Transfer of Glial Cell Line-Derived NeurotrophicFactor (GDNF) . . . . .

12. In Vitro Methods Experiment using rat strial slices Dopamine stimulated adenyl cyclase activity Culture of Substantia Nigra Inhibitionof Apoptosis in NeuroblastomaSH-SY5Y Cells . Radioligand binindg studies for D1 and D2 dopamine receptor. In vitro neuroprotective efficacy

13. PURPOSE AND RATIONALE The muscarinic agonists tremorine and oxotremorine induce parkinsonism-like signs such as tremor, ataxia, spasticity, salivation, lacrimation and hypothermia. These signs are antagonized by anticholinergic drugs. 1.Tremorine and oxotremorine antagonism

14. PROCEDURE

15. TREMOUR SCORE ABSENT 0 SLIGHT 1 MEDIUM 2 SEVERE 3 Salivation and lacrimation are scored 15 and 30 min after oxotremorine injection. absent 0 slight 1 medium 2 severe 3

16. 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.  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

17. MODIFICATIONS OF THE METHOD Matthews and Chiou (1979) developed a method for quantifying resting tremors in a rat model of limb dyskinesias . The model involved permanent cannulation of the caudate nucleus for the introduction of carbachol . Tremors were quantified with a small transducer and an electronic data collecting system. The system allows the construction of dose–response curves for tremor inhibition by potential antiparkinsonism drugs. Johnson et al. (1986) Developed a procedure for quantifying wholebody tremors in mice. Displacement of a free floating platform by animal movement created a change in resistance across a strain gauge. Administration of oxotremorine , 2.5 mg/kg, i.p , produced numerous high-frequency, highintensity peaks within 5 min.

18. Clement and Dyck (1989) Constructed and tested a tremor monitor that quantitates soman - and oxotremorine -induced tremors. The device consisted of a force transducer, from which a plastic beaker was suspended containing a mouse. The signal from the force transducer was fed into a tremor monitor and quantitated using the Applecounter from Columbus Instruments. Coward et al. (1977) Recommended N-carbamoyl-2-(2,6dichlorophenyl) acetamidine hydrochloride (LON1954), a tremorigenic agent, as alternative to oxotremorine for the detection of anti-Parkinson drugs.

19. 2.MPTP Model in animals  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.

20. MPTP model in monkey  Principle: I.V N-MPTP Partial damage to basal ganglia leads to DA precursor L dopa reversed PD like syndrome Requirements :  Animal - Rhesus monkey (5-8 kg).  Drug - N-MPTP up to 10-18 mg/kg i.v. for 5-8 days. Test drug.

21. 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

22.  Evaluation :  The severity is rated by using scale of 0 (normal ) to 17 (max) Observation Scoring (1) Movement Normal 0 Reduced 1 Sleepy 2 (2) Checking movement Present 0 Reduced 1 Absent 2 (3) Attention& blinking Normal 0 Abnormal 1

23. OBESRVATION SCORING (4) Balance & co-ordination Normal 0 Impaired 1 Unstable 2 Falls 3 (5) Vocalization Normal 0 Reduced 1 Absent 2

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

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

26. 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.

27. Reserpine antagonism  Principle : Reserpine induces depletion of central catecholamine stores . The sedative effect can be observed in mice shortly after injection,followed by signs of eyelidptosis , hypokinesia , rigidity, catatonia, and immobility. These phenomena can be antagonized by dopamine agonists.  Requirement s:  Animal : male rats ( Wistar strain 280-300 gm  Drug – Chloral hydrate (350mg/kg i.p .) Reserpine (5mg/kg i.p .)  Apparatus- photocell 3.Reserpine antagonism

28. Male NMRI rats of either sex are taken 20-25g reserpine (5mg/kg i.p .) injected to rats and tested 24hrs later 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 PROCEDURE

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

30. 4. Circling Behavior in Nigrostriatal Lesioned rats Principle : Unilateral lesion of the dopaminergic nigrostriatal pathway in the rat by the neurotoxin 6-hydroxydopamine (6-OHDA) 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 directly acting dopamine agonist,e.g., apomorphine,orthe dopamineprecursor L- dopaisgiven . 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 antiparkinsoniandrugs .

31. Requirements: Animal - Male Wistar rats(200-250gm) Dose - 6-OHDA neurotoxin 6-hydroxydopamine (8µg in 4µl of 0.2mg/ml ascorbic acid in saline ) & test drug Apparatus – Rotameter 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.

32. Procedure: Male Wistar rats rats are taken Rats are anesthetized with Pentobarbital (60mg/kg) Head is placed in stereotaxic device a sagittal cut is made in the skin of the skull, a 2-mm-wide hole is drilled with an electrical trepan drill. .A 30-gaugestainless-steel cannula connected to a Hamilton syringe is aimed at the anterior zona compacta of the substantia nigra

33. After the intracranial injection the wound is closed. The animal is allowed several weeks for recovery and for development of the lesion. A total of 8µg of 6-OHDA in 4γ/l of saline is injected at a rate of 1γ/4min

34. 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. Test compounds are given i.p or s.c. and the animals placed into the circling chambers. Circling is recorded over a 1-h period. 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.

35. 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.

36. 5.Elevation body swing test 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 . Requirements:  Animal - Sprague Dawley rats  Drug - 6-OHDA (8 mg in 4ml 0.9% saline containing 0.02% ascorbic acid). Test drug

37. 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 .)

38. The rat is held about2.5cmfrom the base of its tail and elevated 2.5cm above the surface on which it has been resting. A swing is recorded wheneverthe animal movesits headout of the vertical axis of either side Before attempting another swing, the animal must return to the vertical position for the next swing to be counted. Swings are counted for 60s over four consecutive 15-s segments

39. 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. Srno 15sec 30sec 45sec 60sec LS RS L S RS LS RS LS RS

40. Evaluation :- %LEFT SWING= Σ no. Of swing towards left side Σ L+R %RIGHT SWING = Σ no. Of swing toward right side Σ L+R

41. 6. Skilled paw reaching test 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 . 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)

42. 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

43. 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 45mgsaccharin-flavored pellets are place din each well.

44. 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 4 weeks they are placed in the test boxes once per day for 10–15min. The number of pellets eaten during the test period indicates the rat’s success in grasping and retrieving the pellets; 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 the number of missed pellets remaining at the end of the test on the floor of the side compartmen t indicates a lack of sensorimotor coordination in grasping and retrieving the pellets

45. Lesions The mesotelencephalic system is lesioned by a stereotaxic unilateral injection of 6-OHDA into the medial forebrain bundle under equithesin anesthesia . 6OHDA is injected in a volume of 1.5µl and at a concentration of 4µg/µl of 0.9% saline and 0.01% ascorbic acidtwice over3min viaa 30-gaugestainless steel cannula at the stereotaxic coordinates: Drug Treatment The animals are injected i.p.with the test drug or saline 30min before the unilateral 6-OHDA lesion and 24h there after .

46. 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 x 100 no. of hit Reaching posture • Two point scale • Scored as 0 • Scored as 1

47. 7. Stepping Test in Rats Principle: This model is clinically relevant to unilateral model for parkinsonism akinesia. The 6-OHDA lesion induced marked and longlasting 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

48. Procedure 6-OHDA Lesion Surgery Female Sprague Dawley rats receive two stereotaxic injections of 6-OHDA (3.6µg/µl in 0.2µg/ml ascorbate-saline) into the right ascending mesostriatal dopamine pathway using a 10-µl Hamilton syringe The cannula is left in place for an additional 5min before slowly retracted. The tests monitoring initiation time, stepping time and step length are performed using a wooden ramp with a length of 1m connected to the rat’s home cage.

49. A smooth-surfaced table is used for measuring adjusted steps. During the first 3 days the rats are handled by the experimenter to familiarize them with the experimenter’sgrip . During the subsequent1–2days the rats are trained to run spontaneously up the ramp to the home cage.

50. The stepping test comprises two parts: Each test consists of two tests per day for three consecutive days and the mean of six subtests is calculated.

51. Step length = Length of ramp / no. of steps  Sequence of testing in right paw & followed by left paw testing, repeated twice. Evaluation parameter: Initiation time, Stepping Time, Step length.

52. 8. Gait analysis 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. 9 . Rotenone induced parkinsonism 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 .

54. 10.Transgenic Animal Models ofParkinson’s Disease Purpose and rationale : The most prominent models are related to α- synuclein . The first transgenic mice that express human α- synuclein were generated by Masliah et al. (2000). These mice displayed a progressive accumulation of α synuclein and ubiquitin-immune reactive inclusions in neurons of the neocortex, hippocampus, and substantia nigra . These alterations were associated with a loss of dopaminergic terminals in the basal ganglia and with motor impairments.

55. INVITRO METHOD EXPERIMENT USING RAT STAITAL SLICES : Striatum in brain is primarily affected in parkinsonism.The release of the neurotransmitter like dopamine and acetylcholine in response to test agent serve as a good in vitro marker of its activity. Male spargue dawley rats(150-250g)are decapitated,the skull is opened. Right and left striata are removed & placed in ice-cold krebs solution. The striata is cut into 0.4mm thick slices using a tissue chopper. .

56. The slices are labeled by incubating for 30 min at 37C with [3H]dopamine (5 μ ci/ml) &[14c] choline (2 μ ci/ml)in the presence of 0.15mM pargyline chloride & 0.1mM ascorbic acid. Labeled slices are transferred to superfusion chambers & perfused with krebs solutions at 37c at flow rate of 0.5ml/min The slices are kept floating for 30 min in krebs solution & gassed with 95% o2 & 5% co2 at room temperature After washing & stabilization 5min fraction of superfusate are collected.

57. The perfusion buffer contains 1 μ M nomifensine to inhibit dopamine reuptake & 10 μ M hemicholinium to inhibit choline uptake. The slices are subjected to field strength to the current strength of 10-15mA/cm2 & pulse duration of 2msec at stimulation frequency of 3Hz for 5min. Drugs to be tested are present in the superfusion fluid.

58. The radioactivity in the superfusate samples & in the tissue is determined by liquid scintillation counting. The radiolabelled choline method makes it possible to study Ach release in vitro without inhibiting cholinestrase , thus minimizing auto inhibition of transmitter release caused by accumulation of unhydrolised of Ach.

59. DOPOMINE STUMILATED ADENYLY CYCLASE ACTIVITY Male sprague - dawley (150-250g) are decapitated & right & left striata are removed. Striatal tissue is homogenized by teflon homogenizer in chilled buffer containing 10mM imidazole, 2mM EDTA & 10% sucrose ph 7.3. Homogenate is centrifuged at thousand g for10min & supernatant is recentifuged at 27000g for20min. The pellet obtained is washed twice & suspended in 10mM imidazole, ph 7.3. Membrane protein is determined by bradfords method using bovine serum.

60. Adenylyl cyclase activity is measured by calculating the conversion rate of (32p) ATP to (32p) cAMP . The assay is perform in 250 μ l solution containing imidazole, mgcl2 papaverine dithiothreitol , ATP,GTP,phspocreatine,creatine phosphokinase. The reaction mixture is preincubated at 30c for 5min, the reaction is initiated by adding membrane proteins and incubated for 10min. The reaction is terminated by adding stopping solution( ATP,SDS,cAMP ). Formed (32p) cAMP is separated from (32p) ATP by chromatography.